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Since Virchow triade, it is well established that venous thrombosis is a multifactorial process involving various cellular and plasmatic protagonists. Aspirin antihrombotic efficacy seems not only due to its antiplatelet effects and thromboxane A2 synthesis inhibition. Anti-Platelet Trialists Collaboration metaanalysis stressed in 1994 the interest of aspirin treatment leading to 40% reduction of thrombosis relative risk. Regarding studies heterogeneity and outcomes criteria variety, its use in such context remains a matter of debate. Is the recent publication of PEP trial showing a significant decrease of pulmonary embolism mortality (0.6 versus 0.3%, p = 0.03) able to reinforce aspirin use in venous thrombosis prophylaxis? Were numerous and consecutive criticisms justified? Is there still a potential indication for aspirin in this setting? The experts of the last ACCP consensus conference recommended not to recommend aspirin in venous thrombosis prophylaxis with the highest level of evidence (grade A).

Associated with rheological conditions, multiple factors regarding haemostasis and fibrinolysis contribute to the instability and rupture of atherosclerotic plaque. Various biological parameters are modified during the occurrence of a coronary thrombotic episode, but do these increasingly identified alterations constitute a cause or a direct consequence of this accident? With the increasing identification of markers more and more sensitive but of limited specificity, the relative abundance of studies in the literature contrasts with the paucity of effective assets for everyday clinical practice. In fact, the potential use of these indicators in the evaluation of the pathogenesis of vascular accident, their eventual prognostic value, their predictive nature in therapeutic strategy, and their relevance for patient classification and follow up remain to be established on a wider scale. There is growing interest in the detection of evidence of cellular cooperation or a vascular compartment lesion thanks to the development of new diagnostic tools such as flux cytometry or immunoenzymology. While advances in biology have allowed undeniable clarifications of the physiopathology of coronary accidents and in the management of thrombotic pathology, it must nevertheless be endeavoured to give a practical response to the numerous questions where doubt remains, and to pursue the search for markers or tests clinically relevant.

1: Prostaglandin H synthase-2 (PGHS-2), is an inducible enzyme involved in various inflammatory responses. We established here that interleukin-1beta (IL-1beta) but not tumour necrosis factor-alpha (TNF-alpha) increased its expression in human pulmonary microvascular endothelial cells (HPMEC). However, associated with IL-1beta, TNF-alpha greatly potentiated this enzyme induction. 2: Although unable to induce PGHS-2 expression by itself, TNF-alpha promoted a similar transcription nuclear factor-kappaB (NF-kappaB) activation to IL-1beta. This effect was more pronounced when cells were co-exposed to both cytokines. HPMEC pre-treatment with MG-132, a proteasome inhibitor, prevented NF-kappaB activation as well as more distal signalling response, indicating that NF-kappaB activation is required but not sufficient for PGHS-2 expression. 3: Both IL-1beta and TNF-alpha failed to activate c-Jun NH2-terminal kinase (JNK). In addition, PD98059, a p42/44 mitogen-activated protein kinase (MAPK) phosphorylation inhibitor, did not decrease PGHS-2 expression. However, SB 203580, a p38 MAPK inhibitor, suppressed PGHS-2 induction by IL-1beta alone or combined with TNF-alpha, demonstrating that p38 MAPK but not p42/44 MAPK or JNK cascades are required for PGHS-2 up-regulation. 4: Finally, TNF-alpha, unlike IL-1beta, was unable to promote p38 MAPK phosphorylation, indicating that the failure of TNF-alpha to induce PGHS-2 expression is linked, at least in part, to its inability to activate p38 MAPK signalling pathway. Altogether, these data enhanced our understanding of PGHS-2 regulation in HPMEC and emphasize the heterogeneity of cellular responses to proinflammatory cytokines.

The usefulness of D-dimers determination for the exclusion of deep vein thrombosis (DVT) has been extensively studied. The persistence of high levels of D-dimers has also been suggested as a marker of hypercoagulability in rare studies and might be used to identify patients at risk for recurrent DVT. We have studied the influence of oral anticoagulant treatment in 149 patients, 17 to 84 year-old, with a history of venous thromboembolism; 81 received oral anticoagulant treatment, 68 did not. Patients with known reasons for high level of D-dimers such as cancer were excluded. Thrombophilia was found in 84 patients. D-dimers measurements were performed by ELFA technique using Vidas (bioMérieux, France) analyzer. A significantly lower level of D-dimers was observed in patients under oral anticoagulant compared to patients without this treatment, 197 +/- 134 mug/L versus 399 +/- 239 mug/L, respectively (p < 0.001). A level upper the normal value (500 mug/L) was found in only 3 patients out of 81 receiving an oral anticoagulant treatment as compared with 21 of the 68 patients without treatment. This decrease of D-dimers in patients receiving oral anticoagulants was the same in the different age populations. There was no correlation between INR and D-dimers levels in this study. The clinician should be informed of the decrease of D-dimers in patients treated with anticoagulants. The decrease of D-dimers plasma level during oral anticoagulant treatment suggest that D-dimers concentration in plasma is an indirect marker of reduced clotting activity in vivo.

Background: Perioperative activation of hemostasis could play an important role in the occurrence of postoperative cardiac events. The authors conducted a prospective study to assess platelet function, coagulation, and fibrinolysis status during and after infrarenal aortic surgery., Methods: Seventeen patients were studied. Excluded were patients with preoperative coagulopathies or liver disease, or cardiac or renal insufficiency; patients receiving anticoagulant treatment, antiplatelet agents, nonsteroidal antiinflammatory agents, fresh frozen plasma, or platelet concentrates; and patients undergoing reoperation and septic patients. Blood samples were drawn before induction (T1), 1 h after incision (T2), 1 h after extubation (T3), 24 h postoperatively (T4), 48 h postoperatively (T5), and at day 7 (T6). The following tests were performed: platelet count, platelet aggregation, platelet flow cytometry for CD62 and CD63, usual coagulation tests, thrombin--antithrombin complexes, plasminogen activator inhibitor 1., Results: A significant increase of adenosine diphosphate--induced platelet aggregation was observed postoperatively at T4 and T5. This was not associated with a change of flow cytometry profile. No increase of thrombin--antithrombin complex levels was observed. A higher fibrinogen rate was detected at T5 and T6. Greater amounts of plasminogen activator inhibitor 1 were detected at T3 and T4. Thus, thrombin generation was limited and fibrinolysis was impaired postoperatively. Platelets were not activated in the postoperative period, as shown by flow cytometry, but were prone to be activated, as shown by aggregation studies., Conclusion: The association of more easily activated platelets with a higher fibrinogen rate and a temporary shut down of fibrinolysis during the early postoperative period may indicate an increased thrombotic risk in patients undergoing major vascular surgery.

Heparin-induced thrombocytopenia (HIT), a drug-induced immunohaematological adverse reaction, is a rare but potentially very severe condition. The main problem for this complex syndrome is its recognition and management, which should be as early as possible to avoid the development of life-threatening complications. Most studies have reported heterogeneous populations of patients with other diseases that potentially induce thrombocytopenia. There is no gold standard diagnostic criteria, and we have established a score with anamnestic criteria that allows us to evaluate the likelihood of HIT. In clinical practice, the diagnosis is based on the analysis of clinical features and laboratory tests. Platelet aggregation test (PAT) and an ELISA test (heparin platelet-induced antibodies) are generally performed by expert laboratories to confirm the occurrence of HIT. In our experience, both tests are concordant in the majority of patients. PAT seems to correlate better with the clinical features while ELISA appears more specific. Regarding their limits, both are complementary in the determination of HIT diagnosis coupled to the clinical score system. The treatment often requires a multidisciplinary approach. Danaparoid (Orgaran) or lepirudin (Refludan) are the two alternative treatments for HIT patients with marketing approval. To avoid further exposure to heparin, every HIT patient should carry a written document that confirms the immunoallergy.

Matrix metalloproteinase 2 (MMP2) has been reported to be secreted by collagen-stimulated platelets, and active MMP2 has been shown to play a role in platelet aggregation. It has been demonstrated that MMP2 activation is dependent on the complex (membrane type 1 [MT1]-MMP/tissue inhibitor of MMP2 [TIMP2]) receptor and MMP2. We have investigated human platelets as a possible source of MT1-MMP, and we have studied its role in MMP2 activation and in platelet aggregation. Gelatin zymograms showed the existence of MMP2 at proforms (68 kd) and activated-enzyme forms (62-59 kd) in supernatants of resting and activated platelets, respectively. No gelatinolytic activity was associated with the platelet pellet after aggregation, suggesting a total release of MMP2 during cell activation. By Western blot analysis in nonreduced conditions, MT1-MMP was found on resting platelet membranes in 2 forms-the inactive 45-kd form and an apparent 89-kd form, which totally disappeared under reduced conditions. After platelet degranulation, only the 45-kd form was detected. Reverse transcription-polymerase chain reaction experiments showed the expression in platelets of messenger RNA encoding for MMP2, MT1-MMP, and TIMP2. Flow cytometry analysis showed that MT1-MMP, MMP2, and TIMP2 expressions were enhanced at the activated platelet surface. MMP inhibitors, recombinant TIMP2, and synthetic BB94 inhibited collagen-induced platelet aggregation in a concentration-dependent manner, indicating the role of activated MT1-MMP in the modulation of platelet function. In conclusion, our results demonstrate the expression of the trimolecular complex components (MT1-MMP/TIMP2/MMP2) by blood platelets as well as the ability of MMP inhibitors to modulate the aggregating response.

Two types of thrombocytopenia occur during treatment with heparin: type I and type II heparin-induced thrombocytopenia (HIT). Type I HIT is due to a direct interaction between heparin and platelets. They are asymptomatic, occur early, mild and transitory. Type II HIT which is immunoallergic in nature is the most important complication of this treatment. The thrombocytopenia has a different presentation, acute with a fall in platelets over 30 per cent and is often associated with the occurrence or worsening of a venous or arterial thrombotic episode. The major problems of this secondary type are threefold: its recognition, its confirmation and its management should be as early as possible to avoid the development of often dramatic complications which compromise the prognosis. Laboratory investigations are required by highly reliable specialist laboratories following a careful clinical history. The natural history of the platelet count should enable the difficult diagnosis of HIT to be made more accurately. The treatment of confirmed HIT and/or symptomatic HIT often requires a multidisciplinary approach from a specialized team involving clinicians and hematologists. Two therapies with the benefit of large experience have just been obtained in France with marketing approval (AMM) for the management of HIT: danaparoid (Orgaran) and recombinant hidurin, lepirudin (Refludan). A declaration to the regional drugs monitoring center should not be omitted. In addition, each patient should be given a certificate confirming the immunallergy thus avoiding any further exposure with potentially dramatic consequences.

Myocardial infarction is the result of thrombotic coronary artery occlusion. Although present-day thrombolytics have major value by increasing the frequency of reopening of arteries responsible for myocardial infarction, by preserving myocardial function and, thereby, significantly reduce mortality. Nevertheless, they are subject to the following limitations: 1) excellent arterial partency is only obtained in 50% of cases: 2) reocclusion occurs in 5 to 10% of cases; 3) severe complications such as cerebral haemorrhage are observed in about 0.5% of cases. Therefore, the search to improve thrombolytic agents is intense. This article reports the recent advances in concept and production of new thrombolytic agents. The most recent results concern the production of mutants of T-PA (tissue plasmogen activator). Of these mutants, the reteplase (r-PA) has already received authorization for its commercialisation. Other t-PA mutants under development (phase 3) include TNK-t-PA and lanoteplase. Over the last few years, there has been renewed interest in staphylokinase. The results of the initial clinical trials with this agent have also been reported. Paradoxically, the mode of action of thrombolytic agents has an inherent pro-thrombotic effect. This explains some of the interest for anti-thrombotic agents as an adjuvant treatment of thrombolysis. The initial results of the association of thrombolytics with new glycoprotein IIb/IIIa platelet inhibitors and anti-thrombin agents are reported.

Hereditary thrombophilia is a multifactorial disease which is mono- or plurigenic and its clinical expression is associated with a heterogeneous expression. Factor V (FV) Leiden and FII gene mutations are more frequent than antithrombin, and protein C and S deficiencies. All thrombophilias are not the same. Heterozygous carriers of FV Leiden or FII gene mutation have a weaker risk of venous thrombosis. The mean age at the first episode is older in the former and higher rate of recurrences is observed in the latter. The cosegregation of mutations significantly increases the risk of thrombosis. Both mutations have a geographic and ethnic distribution in relation with a gene founder effect. Clinical expression consists of deep or superficial venous thrombosis with or without pulmonary embolism, thromboses at unusual sites (e.g. cerebral, portal, mesenteric) or with an increased incidence of fetal loss and abortion. A precipitating cause is present in more than 50% of patients. The risk pf arterial thromboses seems to be restricted to some protein S and FII gene mutations. Laboratory diagnosis strategy should be oriented by careful selection of patients and preanalytical variables should be considered. It is highly probable that other unindentified gene mutations are, at least partly, other causes of the heterogeneous expression of hereditary thrombophilia., (Copyright 1999 S. Karger AG, Basel)

The aim of this study was to compare the activated clotting time (ACT) obtained with the Hemochron device and the Heparin Management Test (HMT) on a new automated whole-blood coagulometer, the Thrombolytic Assessment System, in patients undergoing angioplasty. Fifty patients undergoing balloon angioplasty were prospectively enrolled. The mean ACT after a 10,000 unit bolus of heparin was 283 +/- 39 sec at the end of the procedure. The mean HMT after 10,000 units of heparin was 286 +/- 31 sec at the end of the procedure in the same patients. The correlation between the two methods was significant (r = 0.6; P < 0.01). The HMT appears to correlate well with standard values obtained with the Hemochron ACT monitor in patients undergoing percutaneous transluminal coronary angioplasty.

Myocardial infarction is due to thrombotic occlusion of a coronary artery. Current thrombolytic agents have demonstrated their major value by inducing a significant reduction of mortality, but they nevertheless present certain limits: 1) excellent arterial patency is obtained in only about 50% of cases; 2) reocclusions persist in 5 to 10% of cases; 3) very severe complications, such as cerebral haemorrhages, have not disappeared (about 0.5% of cases). There is therefore a large field of action for development of the optimal thrombolytic agent. Research concerning staphylokinase, classified among third generation thrombolytic agents, is therefore situated in this context. Staphylokinase is a protein known to possess profibrinolytic properties for more than four decades, but has been the subject of renewed interest over recent years. This article reviews the characteristics, mechanism of action and fibrinolytic properties of staphylokinase. It recalls the main results obtained in animal models and summarizes the main clinical trials in man.

Staphylokinase which is extracted from Staphylococci has been known for more than 40 years as a profibrinolytic compound. It has been obtained more recently by genetic engineering. Staphylokinase activates plasminogen into plasmin. It is a promising new thrombolytic agent for the treatment of acute myocardial infarction and thromboembolic disease in general. It is a fibrin specific thrombolytic agent which does not decrease plasma fibrinogen level in treated patients. However it is antigenic and the group of Louvain has made much effort to reduce its antigenicity without decreasing its thrombolytic activity. Mutants with a reduced antigenic activity have been recently obtained. These new thrombolytic agents could be superior to currently used drugs. Staphylokinase mutants could have a better benefit/cost ratio than other thrombolytic agents.

Platelets are one source of the group II extracellular form of phospholipase A2 (sPLA2) which is involved in the amplification of local and systemic inflammation. Although sPLA2 protein has been described in human platelets, its presence in human megakaryocytes has not been yet established. We demonstrated in this study that the human erythroleukaemia (HEL) cell line, which has megakaryoblastic features, constitutively expresses sPLA2. Using an anti-rhsPLA2 monoclonal antibody (mAb BA11) and dot-blot detection, we showed that HEL cells and platelets release sPLA2 into incubation medium upon stimulation by thrombin. Similar results were obtained for sPLA2 activity detected by a spectrofluorescence assay. Enzymatic activity was abolished by mAb BA11 and by protamine. In both cell types, although released, the major part of sPLA2 remained in the cell pellet, and was probably adsorbed at non-specific membrane sites. Double labelling experiments using mAb BA11 and an anti-GPIIb antiserum revealed the presence of sPLA2 in human bone-marrow megakaryocytes. The use of reverse transcription-polymerase chain reaction conjugated with hybridization analysis demonstrated the presence of mRNA encoding for sPLA2 in platelets and HEL cells. Expression of sPLA2 in platelets and megakaryocytes at both transcriptional and post-translational levels strongly argues in favour of a megakaryocytic origin of platelet sPLA2 and rules out a role for endocytosis of the enzyme from plasma by circulating platelets.

Heparin-induced thrombocytopenia (HIT) is a drug induced immunohematologic adverse reaction which is a rare but potentially very severe accident. Its diagnosis is important for epidemiologic and drug surveillance studies and in order to decide the most appropriate treatment. Its importance is enhanced since there is no gold standard diagnostic criteria. In clinical practice the diagnosis is based on a group of criteria related to clinical events and laboratory tests. We have established a score based on anamnestic criteria which allowed us to evaluate and compare two different laboratory tests: a platelet aggregation test (PAT) and a test for the detection of heparin dependent antibodies (Heparin Platelet Induced Antibodies or HPIA). The functional test PAT which is commonly used in expert laboratories detects antibodies inducing platelet aggregation in the presence of heparin. The HPIA test more recently developed is an ELISA test which detects antibodies directed at heparin-platelet factor 4 complexes. The relative value of theses two methods for the diagnosis of HIT is not well documented. We have analysed the results of these two tests in 273 consecutive patients with a suspicion of HIT. The results were concordant in 70% of patients. In selecting the patients with the lowest and the highest probability of HIT according to the score, PAT was found a more sensitive and HPIA a more specific test than the other. At low probability PAT is more often positive than HPIA 18% and 9% respectively. No test is 100% reliable, the specificity being limited for both tests since in about 20% of cases one or both tests are negative contrasting with a highly probable HIT. In this last group of patients, PAT was more frequently positive (86%) than HPIA (72%). Both tests are negative in 6% of patients suggesting the existence of presently unknown antigenic targets. Considering a group of 19 patients with a high probability of HIT, we have found antibodies against IL-8 or NAP-2 in only 7 patients. The discrepancy between a HPIA positive and a PAT negative encountered in 8% of patients may be explained by the existence of IgA or IgM immunoglobulins since in contrast to IgG they are unable to promote platelet aggregation via the CD32 platelet membrane receptor. This work suggests than neither test is 100% reliable and that they play a complementary role in the diagnosis of HIT. The potential advantage of using both tests should be confirmed in complementary studies

The exposure of human platelets to prostaglandin H2 analogue (PGH2, U46619) induces homologous desensitization and a concomitant adenylate cyclase (AC) sensitization. We demonstrate the involvement of phospholipase C (PLC) in this enzyme sensitization. Pre-incubation of platelets with neomycin, a PLC activity inhibitor, prevented AC sensitization but not PGH2/thromboxane (Tx)A2 receptor desensitization. PGH2/TxA2 receptor desensitization, although necessary, is not sufficient to induce AC sensitization, since neomycin, which prevents AC sensitization, failed to prevent receptor desensitization. Inositol phosphate formation, determined in parallel, was also inhibited. Interestingly, no guanylate cyclase sensitization was noted, suggesting a specific relationship between PGH2/TxA2 receptor desensitization and AC sensitization. In addition, using alkaline phosphatase, a dephosphorylating enzyme, and the tyrosine kinase inhibitor erbstatin, we examined the role of phosphorylation-dephosphorylation on AC sensitization. Effectively, alkaline phosphatase, which has no effect by itself, enhances the cAMP production triggered by prostacyclin in control but not in desensitized platelets. In contrast, erbstatin failed to modify this synthesis, indicating the non-involvement of tyrosine kinase pathway in this process. Our results indicate that the AC sensitization was mediated by PLC and also suggest the participation of other mechanisms, including phosphorylation-dephosphorylation processes. This specific enzyme sensitization may be relevant for the in vivo modulation of platelet activation, in different thrombotic diseases with an increased TxA2 generation.

Background: A genetic variation of the prothrombin (factor II) gene, a G to A transition at nucleotide position 20210, was recently found in patients with familial thrombophilia (predisposition to venous thrombosis). It seems to be frequent in patients with the factor V Leiden mutation. We report a family with the factor V Leiden and/or the genetic variation of prothrombin in 3 members., Case Report: The patient had repeated episodes of deep vein thromboses starting at the age of 30 during the 4th pregnancy. She is a heterozygous carrier of both the factor V Leiden nutation and the prothrombin mutation 20210 A. She has 4 asymptomatic children, aged 28 to 32 and 3 of them have been explored: one son has the prothrombin mutation, one daughter the factor V Leiden and one has none of them., Discussion: This case report illustrates the polygenic nature of thrombophilia which may explain the heterogeneity of clinical expression observed in isolated congenital abnormalities, especially in factor V Leiden mutation.

Recombinant staphylokinase (RSTA) has been shown to offer promise as a thrombolytic agent. In contrast to streptokinase (SK), few studies have been devoted to possible effects of RSTA on platelets. We have compared the capacity of RSTA and SK to trigger platelet aggregation and to modify ADP (2.5 microM) response in platelet-rich plasma (PRP) of 125 healthy subjects. Thus, exposure of PRP to SK (40 to 50 micrograms/ml) induced platelet aggregation in 6 out of 25 subjects. However, under the same conditions, RSTA failed to induce platelet aggregation in all cases (25 out of 25 subjects). In contrast to RSTA, SK (0.4 to 50 micrograms/ml) greatly reduced ADP-induced platelet aggregation in 12 out of 25 subjects. Preincubation of plasma with SK is associated with a decrease in the fibrinogen concentration. Furthermore, there was a good correlation between SK-induced fibrinogenolysis and SK-induced platelet aggregation defect (r2 = 0.9; p = 0.001). No fibrinogenolysis was observed when different amounts of RSTA (0.4 to 50 micrograms/ml) were incubated in plasma for one min. However, there was a marked decrease in fibrinogen level (about 50%) when the plasma was incubated for five min with a very high concentration of RSTA. SK markedly enhanced the platelet response to ADP in 13 out of 25 subjects. In PRP of 6 out of 25 subjects, SK induces platelet aggregation and potentiates platelet response to ADP, however in PRP of 7 out of 25 subjects, SK caused only the increase of platelet response to ADP. The monoclonal antibody anti-Fc gamma RIIa1, I-3 (2 micrograms/ml), abolished SK-induced platelet aggregation and SK-enhanced ADP-induced platelet aggregation. In all cases (25 out of 25 subjects), RSTA failed to potentiate platelet response to ADP. These findings confirm that RSTA has a lesser fibrinogenolytic ability than SK and suggest its negligible effect on platelet function.

Clinical characteristics of thrombophilia associated with heterozygous mutation of factor V are well characterized. In contrast, they are not well documented in homozygous subjects who are rare. Moreover, estroprogestative intake and pregnancy are important precipitating factors of venous thromboembolism in women with factor V mutation. In order to determine difference in clinical expression between homo and heterozygous subjects, two groups of 22 age matched women were compared. A modified technique for the diagnosis of activated protein C resistance has been used, and its great specificity and sensitivity has been confirmed. In these two small series of patients, a greater severity of clinical profile was not clearly evident. However, recurrences and thrombosis during pregnancy were more frequent in homozygous than in heterozygous women. However, the differences did not reach significance. Molecular markers of hypercoagulable states were not regularly increased even in homozygous untreated patients, but they were more often increased in untreated than in treated patients. In contrast, to other varieties of thrombophilia, homozygous mutation of factor V may be associated with a minor clinical severity suggesting the role of environmental factors and/or still unknown molecular alterations.

We investigated the effects of protamine on the release and the activity of 14 kDa type II phospholipase A2 (sPLA2). Protamine blocks both release and activity of sPLA2 from thrombin-stimulated platelets in a concentration-dependent manner. Heparin, an anionic sulfate polysaccharide which has a high affinity for this enzyme, has no inhibitory effect on sPLA2 by itself but it is able to reverse the inhibitory effect of protamine. The liberation by thrombin of platelet factor 4, an alpha-granule constituent, unlike to that of ATP stored in dense bodies, was suppressed by protamine. Platelet aggregation, determined in parallel, was not affected by protamine. Also, protamine did not inhibit platelet arachidonic acid liberation, which is mainly produced by cytosolic PLA2. The non-proteinaceous polycationic hexadimethrine and acidic protein casein failed to inhibit platelet sPLA2 activity. By contrast, the basic polypeptides poly(L-arginine) and poly(L-lysine) potently inhibited sPLA2 activity, indicating the important role of basic amino acids in the inhibitory effect evoked by protamine. Activities of the human recombinant sPLA2 and the unpurified synovial enzyme of patients with rheumatoid arthritis were also inhibited by the same range of protamine, poly(L-arginine) and poly(L-lysine) concentrations. Our results demonstrate that protamine, unlike heparin, blocks platelet sPLA2 release and exerts a reversible inhibitory effect on its activity, probably through the interaction of basic amino acids with the enzyme.

Activated protein C resistance (APC-R) is a recently defined abnormality of the coagulation system predisposing to the development of a hypercoagulable state. The authors have attempted to evaluate the prevalence and clinical manifestation of APC-R by studying a population of 183 patients with a history of venous thromboembolic episodes. Laboratory evaluation of APC-R was performed using the test initially described by Dahlbäck and colleagues based on the activated partial thromboplastin time (APTT) with the Coatest APC resistance kit (Chromogenix, Sweden) on KC10 coagulometer (Amelung, Germany). These results showed a 13% prevalence of APC-R as demonstrated by an APC ratio below 2.0. The male-to-female ratio was 1:7. Most of the thrombotic episodes were deep venous thromboses (50%).

Exposure to streptokinase (SK) elicits anti-SK antibodies (Abs), which inhibit fibrinolysis and induce platelet aggregation. The mechanism of the latter is not fully understood, although it seems to involve platelet binding by a plasminogen streptokinase and anti-SK ternary complex. Anti-SK Abs were purified by affinity chromatography from serum of patients having received SK for acute myocardial infarction (AMI), and were shown to be of the IgG type. Their effects were studied with (i) human platelets in citrated plasma in the presence of SK or acetylated plasminogen-SK activator complex (APSAC), and (ii) in washed platelets, resuspended in Tyrode buffer after lowering the ionic strength, in the presence of APSAC (which provides both SK and plasminogen). An antibody concentration-response curve was obtained, showing a plateau in the presence of 0.1 mg/ml IgG. By increasing the concentration of APSAC, we obtained a unimodal response curve, the optimal concentration of APSAC being 0.05 U/ml. Aggregation was suppressed by chelating calcium with EDTA, blocking fibrinogen binding by the synthetic peptide Arg-Gly-Asp-Ser (RGDS), and raising intraplatelet cAMP with Iloprost (a prostacyclin analogue). Aggregation required the interaction of the anti-SK Ab Fc domain with the platelet Fc-gamma receptor type II, also known as CD32, since: (i) it was blocked by the monoclonal antibody IV-3 directed against CD32, (ii) it did not occur with F(ab)'2 fragments, which block the response to the intact IgG. The clinical relevance of these platelet-activating anti-SK antibodies remains to be determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Objectives: The frequency of activated protein C resistance is not well established for patients with venous thromboembolic disease. We studied resistance to activated protein C in patients with a past history of deep vein or superficial vein thrombosis., Methods: Activated protein C resistance was measured in 175 patients (37 males, 138 females; mean age 40.9 +/- 13.8 years; range 15-77) who had suffered a venous thromboembolic event more than one month earlier. Exclusion criteria were malignancy, known autoimmune disease or known coagulopathy. A control population of 50 healthy subjects were also tested to establish a normal lower limit (mean ratio minus 2 SD)., Results: The lower limit was established at 2.14. According to this definition, there were 29 thromboembolic patients who were resistant to activated C protein (17%). Two of the subjects considered healthy in the control group were also resistant (4%). There was no difference for age or sex between resistant and non-resistant subjects. Comparing our findings with those reported in the literature confirmed that 3 to 5% of healthy subjects and 15 to 25% of patients with history of venous thromboembolism are resistant to activated C protein., Conclusion: Resistance to activated C protein thus appears as a risk factor for thrombotic events which is comparatively more frequent than other causes of thrombotic disease.