Showing papers in "Thrombosis and Haemostasis in 1990"

Thrombosis and pregnancy in congenital deficiencies in AT III, protein C or protein S: study of 78 women.

Abstract: In patients with congenital deficiency in AT III, PC or PS, the risk of venous thromboembolism is increased and the first episode of thrombosis is frequently observed before the age of 40: pregnancy is often the precipitating condition

Thrombin generation in newborn plasma is critically dependent on the concentration of prothrombin

Abstract: The ability to generate thrombin is decreased and delayed in plasma from the healthy newborn infant compared to the adult. Only 30 to 50% of peak adult thrombin activity can be produced in neonatal plasma. To test whether this observation can be explained by the low neonatal levels of the contact or vitamin K dependent factors, we measured neonatal thrombin generation after raising the concentration of these factors to adult values. We also determined whether the addition of a variety of blood products to neonatal plasma improved thrombin generation. An amidolytic method was used to quantitate intrinsic (APTT) and extrinsic (PT) pathway thrombin generation in defibrinated pooled cord plasma from healthy term infants. Added individually, factors VII, IX, X or the contact factors (CF) failed to alter the rate or the total amount of thrombin generated in neonatal plasma. In contrast, the addition of prothrombin increased the total amount of thrombin generated to above adult values in both the APTT and the PT systems but did not alter the rate of thrombin generation. The rate of thrombin generation in cord plasma shortened after a combination of II, IX, X and CF was added to the APTT system or II, VII and X to the PT system. In both systems, the total amount of thrombin generated was linearly related to the initial prothrombin concentration. Each of fresh frozen plasma, cryoprecipitate, plasma from platelet concentrates, or factor IX concentrate (in amounts used therapeutically) caused an increase in the total amount of thrombin generated which was related to the increase in prothrombin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Two daily subcutaneous injections of fragmin as compared with intravenous standard heparin in the treatment of deep venous thrombosis (DVT).

Abstract: In a prospective, randomized, open study 119 consecutive patients with phlebographically verified deep venous thrombosis (DVT) of the leg (36% distal and 64% proximal) were treated either with a low molecular weight heparin (Fragmin, Kabi-Vitrum) subcutaneously (120 anti-FXa U/kg) twice daily or standard heparin (SH) as continuous intravenous infusion (480 IU kg-1 day-1). The Fragmin doses were adjusted to achieve an anti-FXa activity of 0.2-0.4 U/ml before injection and not greater than 1.5 U/ml 4 h after the morning injection. The SH dose was modified to prolong the APTT 2-3 times. Repeat phlebography after 5-7 days showed improvement in 34/45 patients (76%) in the Fragmin group and in 30/49 patients (61%) in the SH group and progress in 2/45 (4%) and 3/49 (6%), respectively. The mean Marder scores decreased from 18.7 +/- 12.1 to 15.7 +/- 12.7 in the Fragmin group and from 16.9 +/- 12.0 to 14.4 +/- 11.8 in the SH group (ns). Two patients in the SH group and none in the Fragmin group had major bleedings. After 22 +/- 7 months follow up 6 rethromboses had occurred in the SH group and 4 in the Fragmin group. Postthrombotic signs and symptoms were similar in both groups. We conclude that two daily sc Fragmin doses seem as effective and safe as continuous SH in the treatment of DVT of the leg.

Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies.

Abstract: Thrombomodulin, TM, is an endothelial cell membrane protein acting as a cofactor for the activation of plasma protein C. Soluble TM is present in plasma and urine of normal subjects. Enzyme immunoassay, EIA, for human TM was developed using mouse monoclonal antibodies against human placental TM in this paper. We obtained four types of the monoclonal antibodies against human placental TM. EIA sandwich method using three types of the monoclonal antibodies enabled us to measure almost all of 6 and 7 TM subspecies in plasma and urine, respectively, except 1 subspecies, 31 kDa TM. There was no interference from other components of plasma and urine in the assay conditions. Titration curves of purified TM in buffer or in normal plasma were linear within the range from 0.08 to 10 ng/ml. The coefficient of variation at 0.08 ng/ml TM was 4.7%. TM titer with buffer, assayed by this method, was reduced by the addition of thrombin at the final concentration of 20 U/ml, but the titer with plasma was not reduced even at 100 U/ml. These concentrations of thrombin are far larger than those which would be formed in circulation. TM levels in plasma and urine of normal subjects collected in the morning were 35.2 ± 8.32 ng/ml (n = 346) and 111 ± 31.6 ng/ml (n = 33), respectively. TM level in plasma did not differ from the level in serum. Circadian fluctuation of plasma TM was not significant in 10 normal adults, although a tendency of increase in TM excretion to urine was found rather in the day time than the other times. It was concluded that this EIA is reliable for TM assay in human plasma and urine, which will reflect activation or injury of endothelial cells.

Dose-response studies on the effect of n-3 polyunsaturated fatty acids on lipids and haemostasis.

Abstract: We have studied the dose-response effects of dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFA's) on lipids and haemostasis. Ten healthy males were each given 1.3 g, 4 g or 9 g of n-3 PUFA's daily for 6-week periods. Bleeding time, HDL-cholesterol and plasminogen activator inhibitor increased with the dose of n-3 PUFA. Plasma fibrinogen and triglyceride levels were reduced in a dose-dependent fashion. After ingestion of 1.3 g of n-3 PUFA's plasma fibrinogen decreased from 9 to 7 mumol/l and HDL-cholesterol increased from 1.2 to 1.3 mmol/l. The bleeding time was prolonged from 5 to 6.5 min while triglyceride levels decreased from 1.2 to 0.9 mmol/l after ingestion of 4 g of n-3 PUFA's. Dietary supplementation with the highest daily dose (9 g) reduced plasma levels of triglycerides, fibrinogen and von Willebrand factor, while bleeding time, plasminogen activator antigen, plasminogen activator inhibitor and the ratio of HDL-cholesterol to total cholesterol increased.

A comparison of general anesthesia and regional anesthesia as a risk factor for deep vein thrombosis following hip surgery: a critical review.

Abstract: We evaluated the evidence in support of the suggestion that the risk of deep vein thrombosis after hip surgery is lower with regional than with general anesthesia. A literature search was performed to retrieve all articles which reported on the incidence of postoperative thrombosis in both fractured and elective hip surgery. Articles were included if the method of anesthesia used was reported and if they used mandatory venography. Based upon the quality of study design the level of evidence provided by a study was graded. In patients who did not receive prophylaxis there were high level studies in elective and fractured hip surgery. All studies showed a statistically significantly lower incidence of postoperative deep vein thrombosis with regional anesthesia (relative risk reductions of 46-55%). There were no direct comparative studies in patients who received prophylaxis. However, between study comparisons did not show even a trend towards to lower incidence of postoperative thrombosis with regional anesthesia.

Dissociation of platelet activation and spontaneous myocardial ischemia in unstable angina.

Abstract: A dynamic thrombotic process, coronary spasm or both can be responsible for recurrent episodes of transient reduction of coronary blood flow in unstable angina. We have investigated the temporal relationship between episodic platelet activation, as detected by increased urinary excretion of 11-dehydro-TXB2, and spontaneous myocardial ischemia, assessed by continuous electrocardiographic monitoring and recording in 21 patients with unstable angina pectoris. In order to validate measurements of metabolite excretion as a reflection of intracoronary platelet activation, we have also performed repeated urine sampling from 8 patients undergoing PTCA and from 6 patients with peripheral vascular disease. The latter showed a 16% coefficient of variation in 3 consecutive 8-h urine samples. 11-dehydro-TXB2 increased significantly, by up to 15-fold, in the 2.5- to 5.0-h urine collection encompassing PTCA and decreased by greater than 50% during the following 2-h period. Patients with unstable angina were characterized by episodic increases (greater than 2 SD of controls) in metabolite excretion, in successive 6-8 h specimens. Paired measurements of 11-dehydro-TXB2 and 2,3-dinor-TXB2 in 15 urine samples did not reveal evidence of altered metabolic disposition of endogenously released TXB2. A total of 125 ECG ischemic episodes were recorded, of which 64% asymptomatic. We have compared these biochemical and ECG changes in patients randomized to i.v. low-dose aspirin or i.v. isosorbide dinitrate and oral diltiazem. Twenty-five of 56 (i.e. 45%) urine samples obtained in aspirin-free periods showed increased metabolite excretion as compared to 15 of 88 (i.e. 17%) samples collected during aspirin.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin effect on bone density.

Abstract: In order to provide estimates of the risks of symptomatic osteoporosis and reduced bone density in premenopausal women treated with long-term (greater than 1 month) heparin therapy, we evaluated a cohort of 61 consecutive premenopausal women previously treated with long-term heparin (cases) and a group of controls matched for age, parity and duration between the last pregnancy and evaluation. All patients underwent dual photon absorptiometry of the lumbar spine and single photon absorptiometry of the wrist and most cases underwent plain lateral radiography of the thoracolumbar spine in order to exclude silent fractures. Although none of the cases suffered symptomatic fractures (0 of 61, 95% confidence intervals 0.0 to 5.9%), there was a significantly greater proportion of cases than controls with bone density below our pre-defined levels. The long-term implications of our findings are uncertain but because it is possible that the reduction in bone density predisposes women to fractures, this potential risk should be considered when treating women with long-term heparin.

Luminographic detection of von Willebrand factor multimers in agarose gels and on nitrocellulose membranes.

Abstract: Two methods for visualization of vWf multimers were compared with respect to sensitivity and detection of normal vWf and vWd variants IIA, IIB, IIC, IID, IIE, and IIF. Autoradiography and luminography after electrotransfer of vWf multimers onto nitrocellulose showed comparable sensitivity with vWf:Ag detectable after 1:500 dilution of normal plasma. The least sensitive method was luminography in agarose gels with vWf:Ag detectable after 1:300 dilution of normal plasma. No difference existed in the banding patterns of plasmas from patients with variant vWd.

Relationships between euglobulin clot lysis time and the plasma levels of tissue plasminogen activator and plasminogen activator inhibitor 1.

Abstract: The relationships between tissue plasminogen activator (tPA), its fast acting inhibitor (PAI-1) and euglobulin clot lysis time (ELT) were investigated with healthy volunteers' plasma. Turbidimetric clot lysis assay by the microtiter plate reader was utilized for ELT with a slight modification. Both tPA and PAI-1 showed the significant correlation with ELT. tPA had a significantly positive, not negative, correlation with ELT (R = 0.387, p less than 0.001). Higher correlation coefficients (R = 0.580, p less than 0.001 and R = 0.599, p less than 0.001) were obtained between ELT and total PAI-1 or free PAI-1 than tPA or tPA-PAI-1 complex (R = 0.427, p less than 0.001). The positive correlation was also obtained between tPA and PAI-1. These data suggest that PAI-1 is a highly important factor for ELT, especially, the amounts of free PAI-1 being the key factor to determine the ELT, which can represent the potential activity of the fibrinolytic system.

Neutralization of enoxaparine-induced bleeding by protamine sulfate.

Abstract: It has been suggested that protamine sulfate is a poor antidote for the bleeding side-effects of low molecular weight heparins (LMWHs) in vivo, since protamine sulfate does not completely neutralize the anti-factor Xa activity of LMWHs in vitro or ex vivo. Therefore, we performed experiments to compare directly the abilities of protamine sulfate to neutralize the anticoagulant activities of the LMWH, enoxaparine, and unfractionated heparin ex vivo, with its ability to neutralize the bleeding side-effects of both compounds in vivo. Bleeding was measured as the amount of blood lost from 5 cuts made in rabbits ears before and after treatment with enoxaparine or unfractionated heparin +/- protamine sulfate. Plasma anti-factor Xa and anti-thrombin activities ex vivo, were measured chromogenically. Doses of 400 and 1,500 anti-factor Xa U/kg of heparin and enoxaparine, respectively, were required to enhance blood loss to the same extent. Protamine sulfate completely neutralized blood loss induced by both compounds, but did not neutralize the anti-factor Xa nor antithrombin activities ex vivo. We conclude that protamine sulfate is an effective antidote for the bleeding side-effects of enoxaparine and unfractionated heparin, despite its inability to completely neutralize their anticoagulant activities.

The platelet count in carcinoma of the lung and colon.

Abstract: Platelet counts were evaluated in 714 patients with advanced non-small cell lung cancer (N-SCLC), small cell carcinoma of the lung (SCCL), and colon cancer entered to a clinical trial. Patients had not received prior chemotherapy. Platelet counts were not different in patients who had received radiation therapy prior to entry to the study. In comparison to the other tumor types, patients with N-SCLC demonstrated an increased prevalence of thrombocytosis (counts greater than 400,000/mm3), higher platelet counts at the time of entry to the study, higher over all mean platelet counts, relative preservation of high platelet levels during disease progression, and no relationship between platelet numbers and the amount of chemotherapy given. By contrast, platelet counts in patients with SCCL were negatively correlated with the absolute amount of cyclophosphamide and adriamycin given, and declined most dramatically with disease progression and death. Platelet numbers did not correlate with fibrinopeptide A or fibrin split product levels suggesting that disseminated intravascular coagulation or fibrinolysis may have had less influence on platelet numbers than certain other factors. By contrast, significant correlations were found for all three tumor types between platelet numbers and other indicators of bone marrow function including anemia, total leukocyte count, and absolute neutrophil count; and the fibrinogen level. Based upon these findings, we postulate that the host response to malignancy, possibly in the form of production of bone marrow-stimulating cytokines, may play a prominent role in regulation of platelet counts in these (and perhaps other) neoplasms, and that a particularly prominent and persistent degree of marrow stimulation exists in patients with N-SCLC.

Crossreactivity of antibodies directed against cardiolipin, DNA, endothelial cells and blood platelets.

Abstract: The interrelationship of antibodies directed against cardiolipin (CL), double stranded DNA (dsDNA), endothelial cells (EC) and blood platelets was investigated. IgG fractions, reactive with these antigens, were isolated from the plasmas from 8 patients with systemic lupus erythematosus and tested for crossreactivity with anionic phospholipids (CL, phosphatidylserine, phosphatidylinositol), zwitterionic phospholipids (phosphatidylethanolamine, phosphatidylcholine), dsDNA, EC and platelets by enzyme-linked immunosorbent assays and for lupus anticoagulant (LAC) activity with a coagulation assay. Our results demonstrate the frequent occurrence of crossreactivity between antibodies to anionic phospholipids, EC and platelets. Crossreactivities between these antibodies and antibodies to dsDNA or zwitterionic phospholipids are exceptional. LAC activity was found in the anti-CL, anti-EC and anti-platelet fractions of only one patient. These findings support the hypothesis that subpopulations of antibodies directed against negatively charged phospholipids can bind to EC and blood platelets, which may have implications for the pathogenetic potential of antiphospholipid antibodies.

Localization of von Willebrand factor and thrombin-interactive domains on human platelet glycoprotein Ib.

Abstract: Platelet membrane glycoprotein Ib (GPIb) functions as receptors for thrombin and von Willebrand factor (vWF) in the presence of ristocetin. To precisely locate the domains on GPIb interacting with vWF and thrombin, we prepared several peptides that have amino acid sequences analogous to that of the GPIb alpha-chain and examined their effects on ristocetin-induced (vWF-dependent) and thrombin-induced platelet aggregations. A peptide extending from residues Asp235 to Lys262 showed the strongest inhibitory effect on ristocetin-induced platelet agglutination, and a group of overlapping peptides composed of 24-28 amino acid residues representing sequences extending from Phe216 to Asp274 was found to inhibit platelet aggregation induced by thrombin. Other peptides did not inhibit platelet aggregations. Moreover, the binding to platelets of the monoclonal anti-GPIb antibody (TM60) which had been shown to inhibit both ristocetin- and thrombin-induced platelet aggregations was strongly inhibited by a peptide extending from Asp249 to Asp274. These data demonstrate that the vWF-binding domain exists in a small region between residues Asp235 and Lys262; the thrombin-interacting domain, in contrast, is located between residues Phe216 and Ala274, with a possible center of interaction in the sequence from Phe216 to Thr240 on the GPIb alpha-chain, and thrombin binding requires a relatively strict conformation in this domain.

Hereditary Protein S deficiency in young adults with arterial occlusive disease

Abstract: Protein S is the vitamin K dependent cofactor of activated protein C. It has an important role in the regulation of blood coagulation and fibrinolysis. Hereditary protein S deficiency is associated with familial venous thrombophilia. Since a few patients with arterial occlusions have been reported to be protein S deficient, it is speculated that hereditary protein S deficiency may be also a risk factor for the development of arterial thrombosis. In a group of 37 consecutive patients with arterial occlusive disease presenting before the age of 45, three patients were found heterozygous for hereditary protein S deficiency. None of the patients had a protein C deficiency or an antithrombin III deficiency. Family investigations showed a clear association between the hereditary deficiency and venous thrombosis, but a relation between the deficiency and arterial thrombosis was less obvious. A review of previous literature on patients with arterial thrombosis and protein S deficiency revealed that more extensive studies are needed to demonstrate whether or not hereditary protein S deficiency is a risk factor for the development of arterial thrombosis.

Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects.

Abstract: Although protein C (PC) and activated protein C (APC) have been postulated to be useful for treating patients with thrombosis, their critical effect remains to be studied in human subjects. To examine whether purified PC or APC are useful for treating patients with thrombosis without showing any adverse effect, we studied effects on coagulation and fibrinolysis in normal human subjects. When highly purified human PC was administered intravenously to healthy subjects, plasma levels of immunoreactive PC decreased with a half-life of 10.9 h. Intravenously administered APC decreased with a half-life of 23 min as measured by prolongation of activated partial thromboplastin time (APTT). However, 1.7 h was obtained for the plasma half-life of APC when it was measured immunologically. These findings suggested that a significant fraction of the administered APC was rapidly inhibited by plasma inhibitor. Upon administration of APC, APTT was prolonged and plasma levels of clotting factor VIII (F-VIII) decreased transiently as measured by clotting assay. However, when determined by a chromogenic assay method in which 120-fold diluted plasma samples were used, plasma levels of F-VIII remained unchanged. Plasma levels of F-V did not decrease after APC administration. These findings suggested that prolongation of APTT and apparent decrease in plasma F-VIII clotting activity might be due to the in vitro-effect of APC present in plasma samples used. Diurnal fluctuation of plasminogen activator inhibitor in normal subject was not affected by administration of APC. Thus, PC or APC seems to function selectively at the site of thrombin-formation without lowering plasma levels of coagulation factors.

Lack of platelet response to collagen associated with an autoantibody against glycoprotein Ia: a novel cause of acquired qualitative platelet dysfunction.

Abstract: Platelets from a patient with an acquired hemorrhagic disorder had a severely impaired response to collagen, whereas platelet aggregation to other agonists and coagulations tests were normal. No abnormalities of the patient's platelet membrane glycoproteins (GP) were seen. Treatment of the patient with immunosuppressive agents temporarily improved both the bleeding tendency and the collagen responsiveness of the platelets. An IgG was found to be present in the plasma, directed against a protein comigrating with GPIa, and coadsorbing with GPIa to insoluble collagen fibers in a Mg2+(-)dependent manner. Furthermore, GPIa was recognized by the patient's antibody when affinity-purified GPIa-IIa was used as antigen. Finally, the GPIa-IIa complex was immunoprecipitated from a platelet lysate by patient's plasma. In addition, purified platelet specific IgG's from the patient inhibited aggregation of normal platelets induced by collagen or by wheat germ agglutinin. We conclude that the lack of response to collagen of the patient's platelets may well be due to the presence of an autoantibody against GPIa.

Effects of low molecular weight heparin and unfragmented heparin on induction of osteoporosis in rats.

Abstract: A comparison between the effect of low molecular weight heparin (LMWH) and unfragmented heparin (UH) on induction of osteoporosis was made in 60 rats treated with either UH (2 IU/g bw), LMWH in 2 doses (2 XaI U/g or 0.4 XaI U/g) or placebo (saline) for 34 days. Studied variables were: bone mineral mass in femora; fragility of humera; zinc and calcium levels in serum and bone ash and albumin in plasma. A significant reduction in bone mineral mass was found in all heparin-treated rats. There was no difference between UH and LMWH in this respect. The effect was dose-dependent in LMWH-treated animals. The zinc contents in bone ash were decreased in all heparin-treated rats as compared with controls. No recognizable pattern was seen in alterations of zinc or calcium in serum. The fragility of the humera, tested as breaking strength did not differ between treatment groups and controls. In conclusion, if dosed according to similar factor Xa inhibitory activities, LMWH induces osteoporosis to the same extent as UH and in a dose-dependent manner. The zinc content in bone ash was decreased after heparin treatment, irrespective of type of heparin given.

Monoclonal antibodies specific for a conformationally altered state of fibrinogen.

Abstract: Four monoclonal antibodies were selected by their ability to discriminate surface-bound from soluble fibrinogen. These antibodies reacted with insolubilized fibrinogen but not other immobilized proteins and their reaction with surface-bound fibrinogen was not diminished by a 100-fold excess of soluble fibrinogen. The antibodies reacted with the same or spatially proximal epitopes, and the recognized epitope(s) resided within the gamma chain segment of the D domain of fibrinogen. Fab fragments of the antibodies inhibited fibrin polymerization in a dose dependent manner, suggesting that the epitope(s) was also exposed by the conversion of fibrinogen to fibrin. These data indicate that adsorption of fibrinogen onto a surface induces conformational changes and that similar changes are also evoked when fibrinogen is converted into fibrin.

The comparative effects of recombinant hirudin (CGP 39393) and standard heparin on thrombus growth in rabbits.

Abstract: The aim of this study was to compare the ability of standard heparin and recombinant (r-)hirudin, a specific inhibitor of thrombin, to inhibit thrombus growth in a rabbit jugular vein model. Doses of standard heparin and r-hirudin equivalent in prolonging the aPTT were first identified. The ability of these doses to inhibit 125I-fibrin accretion onto preexisting thrombi was then evaluated. 0.5 and 0.75 mg/kg of standard heparin and 0.8 and 1.25 mg/kg of r-hirudin infused over 3 h produced a mean prolongation of the aPTT of 1.5 and 2 times, respectively. In saline treated rabbits 62 +/- 7 micrograms of 125I-fibrin were accreted on the pre-formed thrombi. The lower doses of standard heparin and r-hirudin produced a 125I-fibrin accretion of 44 +/- 5 and 25 +/- 4 micrograms, respectively (p less than 0.01). The two higher doses of standard heparin and r-hirudin produced a 125I-fibrin accretion of 34 +/- 4 and 17 +/- 3 micrograms, respectively (p less than 0.01). The increase in the dose of standard heparin up to 2.5 mg/kg produced a 125I-fibrin accretion of 26 +/- 3 micrograms a 58% reduction when compared with saline. The increase in the dose of r-hirudin up to 5 mg/kg produced a 125I-fibrin accretion of 12 +/- 2 micrograms, an 81% reduction when compared with saline. No further inhibition was observed when the doses of both agents were further increased. We conclude that doses of standard heparin and r-hirudin equivalent in prolonging the aPTT have a different effect on thrombus growth inhibition, r-hirudin being twice as effective as standard heparin. Exclusive inhibition of thrombin without any other inhibiting effect on blood coagulation appears to be sufficient to inhibit thrombus growth. Our results seem to be promising in view of a clinical evaluation of r-hirudin.

D-dimer and TAT measurement in patients with deep venous thrombosis: utility in diagnosis and judgement of anticoagulant treatment effectiveness.

Abstract: The plasma levels of thrombin-antithrombin III-complexes (TAT) and the fibrin split product D-Dimer were measured in 39 patients with phlebographically proven acute DVT: 34 patients had proximal DVT, 5 had calf DVT. The sensitivity of D-Dimer and TAT measurements in the diagnosis of proximal DVT was found to be dependent on the duration of symptoms: 0 to 7 days (n = 27): elevated D-Dimer levels (greater than 120 ng/ml) = 1, D-Dimer Latex test positive (greater than 500 ng/ml) = 1, elevated TAT levels (greater than 6 ng/ml) = 0.88. Eight to 14 days (n = 7): elevated D-Dimer levels = 1, D-Dimer Latex test positive = 0.33, elevated TAT levels = 0.66; specificity: elevated D-Dimer: 0.48, D-Dimer Latex test: 1, elevated TAT: 0.76. Calf DVT patients (n = 5) had elevated D-Dimer levels, negative Latex tests and 3 of them had normal TAT values. Hemostatic and fibrinolytic parameters were also determined in 13 patients during heparin treatment of proximal DVT. Elevated D-Dimer and TAT levels rapidly decreased after initiation of anticoagulant therapy. In 2 of 13 patients a marked increase in D-Dimer and TAT levels was observed in periods of ineffective heparinization, documented by normal or only slightly prolonged thrombin clotting times. We conclude from our results that 1) D-Dimer EIA measurement, in contrast to TAT measurement, shows a very high sensitivity in the diagnosis of DVT, 2) due to low specificity this test can only be used to exclude thrombosis in patients with suspected DVT, and 3) the determination of the plasma levels of D-Dimer and TAT may be useful for judging the effect of anticoagulant treatment on thrombotic processes.

Formation and persistence of procoagulant and fibrinolytic activities in circulation after strenuous physical exercise

Abstract: Seven healthy male volunteers were subjected to exercise of short (STR; 1.7 km), middle (MTR; 4.8 km) and long (LTR; 10.5 km) term runs at a speed close to maximal capacity. Blood samples were drawn before, immediately after exercise and at intervals over the next 10 h. FVIIIR:Ag (von Willebrand factor) rose 2.2-3.2 fold and persisted at higher levels than baseline during the observation time. A spontaneous drop in FVII (p less than 0.03) was found immediately after STR (13.5 +/- 2.5%) and LTR (18.3 +/- 2.4%), whereas only a minor decrease (7.5 +/- 6.5%) occurred in MTR. The procoagulant activity of monocytes isolated from whole blood exposed to LPS showed a striking enhancement in STR and MTR. An immediate enhancement in fibrinolytic activity was found in all groups (p less than 0.03) assessed by increased plasma levels of t-PA and shortened whole blood clot lysis time (WBCLT). The transient shortening of WBCLT was succeeded by a tendency to prolongation of the lysis time. A 45-year old male differed markedly from the others by demonstrating an extreme and consistent prolongation of WBCLT. Thus, it has been speculated that strenuous exercise possibly makes a subject more susceptible to a thrombotic event.

Ristocetin and botrocetin involve two distinct domains of von Willebrand factor for binding to platelet membrane glycoprotein Ib.

Abstract: We have evidence that ristocetin and botrocetin mediate binding of von Willebrand Factor (vWF) to platelet glycoprotein lb (GPIb) through two distinct domains on the vWF molecule. This was established by using monoclonal antibodies (MAbs) to vWF and synthetic peptides derived from the sequence of vWF. MAb 322 and MAb NMC/vW 4 both recognize native vWF as well as fragments containing the GPIb-binding domain of vWF, obtained with the following enzymes: trypsin (116 kDa), V-8 pro tease (Spill, 320 kDa) and V-8 protease plus subtilisin (33-28 kDa). Nevertheless, the lack of reciprocal displacement between the two MAbs in experiments of competitive inhibition for binding to vWF demonstrate that their respective epitopes are separate. Both MAbs inhibit 125I-vWF binding to platelet membrane GPIb and vWF-dependent platelet agglutination induced by ristocetin. However, only MAb NMC/vW4 inhibits these functions in the presence of botrocetin and when ristocetin-induced platelet agglutination is inhibited by MAb 322, botrocetin is still able to restore the agglutination. The involvement of two distinct domains of vWF for binding to GPIb in the presence of ristocetin or botrocetin was confirmed in experiments of binding of 125I-vWF to platelets using as competitor synthetic peptides corresponding to the GPIb binding domain of vWF (Cys 474 to Pro 488 and Ser 692 to Pro 708). At a final concentration of 2.5 mM both peptides inhibit more than 90% of the binding of vWF to ristocetin-treated platelets but are unable to modify this binding in the presence of botrocetin. In conclusion our data suggest that botrocetin and ristocetin involve distinct sites on vWF for binding to GPIb.

Effects of gemfibrozil on lipids and haemostasis after myocardial infarction.

Abstract: The effects of gemfibrozil on haemostatic variables were studied in 43 survivors of myocardial infarction with serum triglycerides (TG) greater than or equal to 2 mmol/l 2 weeks prior to randomization. The study was double-blind, placebo-controlled and stratified for chronic betablockade. Twenty-two individuals were given gemfibrozil 600 mg twice daily and 21 individuals received matching placebo. After 8 weeks the TG level was unchanged in the placebo group, whereas a 44% reduction was noted in the gemfibrozil group (p less than 0.001). Fibrinogen increased in both groups, while bleeding time and platelet count were unchanged. Clotting factor VII-phospholipid complex decreased in both groups, but the change was more marked and attained statistical significance only in the gemfibrozil group (60% reduction, p less than 0.01). By DDAVP-stimulated D-Dimer agglutination test 8 in 21 patients in the placebo group (38%) still had reduced fibronolytic capacity versus none in the gemfibrozil group (p = 0.001). Thus, in this study, gemfibrozil improved reduced fibrinolytic capacity and may have reduced hypercoagulability by lowering the clotting factor VII-phospholipid complex.

Clearance of tissue plasminogen activator by mannose and galactose receptors in the liver.

Abstract: The mechanism of uptake of radio-iodinated tissue plasminogen activator (125I-t-PA) was studied in rats. When trace amounts of 125I-t-PA were injected alone, the clearance followed a biphasic pattern in which 65% and 35% were cleared with α- and β-kinetics (t1/2 (α) = 0.6 min, and t1/2 (β) = 6.4 min), respectively. Coinjection with excess unlabelled t-PA or inannan changed the uptake kinetics to the muiiupliasic β-elimination pattern. Mannosylated albumin and ovalbumin, both of which bind to the hepatic mannose receptor, reduced the proportion of t-PA cleared with t1/2 (α) to 48% and 21%, respectively. A corresponding increase in the β-elimination ot t-PA was observed. The t1/2 (α) and t1/2 (β) were unchanged. Studies on the eleaiaucc of 125I-ovalbumin also showed a biphasic elimination with an initial rapid phase, t1/2 (α), accounting for only 39% of the clearance of ovalbumin, as compared to 65% in the case of t PA. Macromolecules with affinity for the galactose-receptor only, such as asialofetuin, or galactosylated albumin, did not significantly affect the clearance kinetics at the concentrations used. Asialoorosomucoid, which also carries galactosyl residues in the terminal position, reduced somewhat (from 65% to 48%) the proportion cleared with α-kinetics. Very high concentrations of galactose and N-acetyl-galactosamine, which are also known to compete for binding to the galactose receptor, lowered the proportion of t-PA cleared in the late β-phase (reduced from 35% to 26% with galactose and to 19% with N-acetyl-galactosamine). To determine the hepatocellular site of uptake of t-PA, the protein was conjugated with 125I-labelled tyramine cellobiose (125I-TC) and injected intravenously (i.v.). This adduct is nonbiodegradable, and is trapped intralysosomally after endocytosis. I.v. injection of 125I-TC-t-PA and subsequent isolation of the liver cells showed that the Kupffer cells (KC), liver endothelial cells (LEC) and parenchymal cells (PC) contained 11%, 44% and 45%, respectively, of the radioactive label recovered in liver (hepatic uptake 80% of injected dose). The in vivo uptake per cell was about three times higher in KC and LEC than in PC. Injection of 125I-TC-t-PA together with mannose inhibited uptake in LEC and increased uptake in PC. Conversely, co-injection with galactose inhibited the uptake of 125I-TC-t-PA in PC and increased the uptake in LEC. Co-injection with excess amounts of unlabelled t-PA shifted the site of uptake from LEC to PC and changed the clearance kinetics to a monophasic β-elimination. The inhibitors used had only marginal effects on the uptake of 125I-TC-t-PA in KC. Although significant amounts of label were recovered in KC, the total size of the population of these cells is relatively small, so that the main hepatic uptake of 125I-TC-t-PA was in LEC and PC. In conclusion, the elimination of t-PA from the blood by the liver is strongly dependent on the structure of its carbohydrate side chains. The main cellular sites of clearance are LEC (via mannose receptors), and PC (via galactose receptors and an unsaturable noncarbohydrate uptake mechanism).

The dependence of the International Sensitivity Index on the coagulometer used to perform the prothrombin time.

Abstract: This study was designed to detect any effect that different types of coagulation instrument may have on the International Sensitivity Index (ISI) of a thromboplastin. Manufacturers of commercial thromboplastins now calibrate their reagents against the World Health Organization international reference preparation to assign them an ISI. This enables the prothrombin time (PT) estimated with that reagent to be expressed as an International Normalised Ratio (INR). One batch of Thromborel S was calibrated against the Australasian Reference Thromboplastin (ART). The Thromborel S was used on three photo-optical instruments, the Automated Coagulation Laboratory (ACL) (Instrumentation Laboratory), the Cobas Fibro (Roche), and the Coag-a-Pet (General Diagnostics). PTs using ART were performed manually using the reference method. The ISIs calibrated in our laboratory when the ACL and Cobas Fibro were used were not significantly different at the 95% level, being 1.102 +/- 0.018 and 1.134 +/- 0.022 respectively. The ISI with the Coag-a-Pet of 1.223 +/- 0.023 was significantly different to that of the ACL and the Cobas Fibro at the 95% level. The flowcharts for a computer program to perform the necessary calculations are provided. The program allows for the entry and editing of data from the calibration procedure, and provides a mean normal PT and normal range, the ISI and 95% confidence limits of the calibration, and a chart for the conversion of the test PTs to INRs. The authors have made available an IBM compatible program for the calibration of thromboplastins.

Subcutaneous vs intravenous heparin in the treatment of deep venous thrombosis : a randomized clinical trial

Abstract: 271 patients with acute symptomatic deep venous thrombosis of lower limbs, confirmed by strain-gauge plethysmography and/or venography, were randomly assigned to receive intermittent subcutaneous heparin calcium or heparin sodium by continuous intravenous infusion for 6-10 days. Heparin dosage was adjusted to maintain activated partial thromboplastin time values (Thrombofax reagent) at 1.3-1.9 times the basal ones. Strain-gauge plethysmography was repeated at the end of heparin treatment, and evaluation of therapy was performed by comparing the indexes of venous hemodynamics and by assessing the incidence of pulmonary embolism and of bleeding complications. In the intravenous group, Maximal Venous Outflow (MVO) increased from 20.8 +/- 12.8 to 28.4 +/- 17.5 ml/min per 100 ml of tissue and Venous Capacitance (VC) from 1.39 +/- 0.92 to 1.94 +/- 1.0 ml/100 ml of tissue (mean +/- SD). In the subcutaneous group, MVO increased from 21.0 +/- 12.7 to 27.5 +/- 18.1 and VC from 1.60 +/- 0.86 to 2.06 +/- 1.0. The median improvement of MVO and VC were 22% and 36% respectively in the IV group and 20% and 24% in the SC group. Clinical pulmonary embolism occurred in 2 patients in the intravenous group (1 fatal) and in 4 in the subcutaneous group (1 fatal). 9 major bleeding complications occurred in the intravenous group (1 fatal) and 5 in the subcutaneous group (1 fatal). The differences were not significant at the statistical analysis. The results suggest that subcutaneous intermittent heparin has a comparable efficacy to continuous intravenous heparin in the treatment of deep venous thrombosis. To the same conclusion points an overview of the seven randomized trials which compared these treatment modalities.

Enhanced effective fibrinolysis following the neutralization of heparin in open heart surgery increases the risk of post-surgical bleeding.

Abstract: The tissue-type plasminogen activator related fibrinolytic system was studied in 24 patients undergoing cardiopulmonary bypass surgery The degradation of fibrinogen and fibrin was followed during and after surgery by means of new sensitive and specific assays and the changes were related to the blood loss measured in the chest tube drain during the first 24 postoperative hours Although tissue-type plasminogen activator was significantly released into the circulation during the period of extracorporeal circulation (p less than 001), constantly low levels of fibrinogen degradation products indicated that a systemic generation of plasmin could be controlled by the naturally occurring inhibitors Following extracorporeal circulation heparin was neutralized by protamine chloride, and in relation to the subsequent generation of fibrin, there was a short period with increased concentrations of fibrinogen degradation products (p less than 001) and a prolonged period of degradation of cross-linked fibrin, as detected by increased concentrations of D-Dimer until 24 h after surgery (p less than 001) Patients with a higher than the median blood loss (520 ml) in the chest tube drain had a significantly higher increase of D-Dimer than patients with a lower than the median blood loss (p less than 005) We conclude that the incorporation of tissue-type plasminogen activator into fibrin and the in situ activation of plasminogen enhance local fibrinolysis, thereby increasing the risk of bleeding in patients undergoing open heart surgery

Human tumor procoagulants: registry of the Subcommittee on Haemostasis and Malignancy of the Scientific and Standardization Committee, International Society on Thrombosis and Haemostasis.

Abstract: The present communication represents the first of two planned papers describing the properties of tumor procoagulants : a review of experimental animal tumors