Haemostatic changes in urogenital schistosomiasis haematobium: a case-control study in Gabonese schoolchildren.

In many tropical areas schistosomiasis is a major health problem causing hepatosplenic, intestinal or urogenital complaints. Hepatosplenic schistosomiasis mansoni is also characterized by blood coagulation abnormalities. Liver pathology plays a role in the development of haemostatic changes and the parasitic infection may directly affect coagulation. However, these contributing factors cannot be studied separately in hepatosplenic schistosomiasis infections. This pilot study provides insight in haemostatic changes in urinary schistosomiasis by studying coagulation parameters in schistosomiasis haematobium-infected Gabonese schoolchildren. Selection on urinary schistosomiasis patients without hepatosplenic complaints allows for the investigation of the direct effects of the parasite on haemostasis. Levels of von Willebrand Factor (VWF) antigen, active VWF and osteoprotegerin were elevated, indicating inflammation-mediated endothelial activation. In contrast to hepatosplenic schistosomiasis, thrombin-antithrombin complex and D-dimer levels were not affected. Despite its small sample size, this study clearly indicates that Schistosoma haematobium directly alters the activation status of the endothelium, without initiation of coagulation.

Morbidity and mortality in the antiphospholipid syndrome during a 10-year period: a multicentre prospective study of 1000 patients.

OBJECTIVES: To assess the prevalence of the main causes of morbi-mortality in the antiphospholipid syndrome (APS) during a 10-year-follow-up period and to compare the frequency of early manifestations with those that appeared later. METHODS: In 1999, we started an observational study of 1000 APS patients from 13 European countries. All had medical histories documented when entered into the study and were followed prospectively during the ensuing 10 years. RESULTS: 53.1% of the patients had primary APS, 36.2% had APS associated with systemic lupus erythematosus and 10.7% APS associated with other diseases. Thrombotic events appeared in 166 (16.6%) patients during the first 5-year period and in 115 (14.4%) during the second 5-year period. The most common events were strokes, transient ischaemic attacks, deep vein thromboses and pulmonary embolism. 127 (15.5%) women became pregnant (188 pregnancies) and 72.9% of pregnancies succeeded in having one or more live births. The most common obstetric complication was early pregnancy loss (16.5% of the pregnancies). Intrauterine growth restriction (26.3% of the total live births) and prematurity (48.2%) were the most frequent fetal morbidities. 93 (9.3%) patients died and the most frequent causes of death were severe thrombosis (36.5%) and infections (26.9%). Nine (0.9%) cases of catastrophic APS occurred and 5 (55.6%) of them died. The survival probability at 10 years was 90.7%. CONCLUSIONS: Patients with APS still develop significant morbidity and mortality despite current treatment. It is imperative to increase the efforts in determining optimal prognostic markers and therapeutic measures to prevent these complications.

Factor VIII concentrate infusion in patients with haemophilia results in decreased von Willebrand factor and ADAMTS-13 activity.

The effects of coagulation factor concentrate infusion on restoring secondary haemostasis in patients with haemophilia are obvious. It is not known whether coagulation factor concentrate infusion affects primary haemostasis or induces an acute inflammatory response. In this study, the influence of a factor VIII (FVIII) concentrate bolus infusion on platelet activation and responsiveness, endothelial activation, and inflammation in adult patients with severe haemophilia A was assessed. VWF showed a mild, but significant decrease 15 min after FVIII infusion (85.02 IU dL(-1)) vs. before infusion (92.04 IU dL(-1) ; P = 0.017), while ADAMTS-13 levels also show a mild but significant decrease from 66.1 ng mL(-1) before infusion, to 53.9 ng mL(-1) (P = 0.012) 15 min after and 50.8 ng mL(-1) (P = 0.050) 60 min after infusion. Platelet P-selectin expression decreased 15 min (33.3 AU) and 60 min (38.7 AU) after infusion compared to before infusion (41.3 AU; P = 0.018 and 0.036). In conclusion, a single infusion of a high dose FVIII concentrate in haemophilia A patients may influence primary haemostasis by decreasing VWF, ADAMTS-13 and the number of circulating activated platelets. These effects possibly occur as a consequence of binding of the infused FVIII to VWF, influencing its processing. When treating severe haemophilia A patients with coagulation concentrate infusion, one should realize this does not merely correct FVIII levels but also may influence primary haemostasis.

Pathophysiology of thrombotic APS: where do we stand?

The antiphospholipid syndrome (APS) is diagnosed when patients with thrombotic complications or foetal losses have elevated levels of antiphospholipid antibodies in their plasmas. The term APS is confusing, because the pathogenic auto-antibodies are not directed against phospholipids but towards a plasma protein, ß(2)-glycoprotein I. For many years the reason why auto-antibodies against ß(2)-glycoprotein I were pro-thrombotic was unclear, because man and mice deficient in ß(2)-glycoprotein I do not express a clear phenotype. Animal models in which passive transfer of patient antibodies into mice resulted in an increased thrombotic response have provided novel insights in the importance of this protein in the pathology of APS.

Thrombogenicity of a new injectable biocompatible elastomer for aneurysm exclusion, compared to expanded polytetrafluoroethylene in a human ex vivo model.

OBJECTIVES: Customized aortic repair (CAR) is a new concept for endovascular aortic aneurysm repair in which a non-polymerised elastomer is injected to fill the aneurysm sac around a balloon catheter. Amongst other variables, the thrombogenicity of the elastomer should be tested, before further clinical experiments can take place. The aim of this human ex vivo study was to measure the thrombogenicity of the elastomer and to compare it to expanded polytetrafluoroethylene (ePTFE). DESIGN AND MATERIALS: In a validated ex vivo model, non-anticoagulated blood was drawn from the antecubital veins of 10 healthy donors with a 19-gauge needle. It was drawn through elastomer tubes and through ePTFE Gore-Tex vascular grafts, both 60 cm long and with an inner diameter of 3 mm. METHODS: Fibrinopeptide A (FPA) and P-selectin expression was measured in blood samples, collected at the end of the grafts. After the experiments, the deposition of platelets and fibrin onto the grafts was visualised by scanning electron microscopy. RESULTS: For these graft types, a progressive increase in FPA production was observed in time. No significant difference was observed between the elastomer and ePTFE grafts (p > 0.05). No increase in P-selectin expression, and thereby no platelet activation, was observed in the perfusate of either grafts (p > 0.05). By scanning electron microscopy, numerous platelet aggregates were observed on the ePTFE grafts, whereas just a few adhered platelets and no aggregates were observed in the elastomer grafts. CONCLUSIONS: The elastomer in its current formulation has a low thrombogenicity, comparable to ePTFE, making it an ideal substance for endovascular aneurysm sac filling. Further research should clarify the feasibility of CAR in vivo.

Antiphospholipid syndrome: laboratory detection, mechanisms of action and treatment.

The antiphospholipid syndrome (APS) identifies a condition at increased risk of vascular occlusion and/or pregnancy complications. Patients are defined as having APS if they have at least one clinical (vascular occlusion and/or pregnancy complications) and one laboratory criterion at the same time. The laboratory criteria that define APS are repeated positivity (confirmed 12 weeks apart) for lupus anticoagulants and/or antibodies targeted against cardiolipin or ß(2) -glycoprotein I immobilized on solid surfaces. Over the years, APS has attracted the interest of many medical specialties. The aim of this review is to provide an update on (i) the laboratory criteria that determine the presence of APS, (ii) how the antibodies increase the risk of vascular occlusion and foetal loss and (iii) the treatment of the related clinical events.

'Criteria' aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, Texas, April 2010.

Current classification criteria for definite antiphospholipid syndrome (APS) mandate the use of one or more of three positive 'standardized' laboratory assays to detect antiphospholipid antibodies (aPL) (viz: anticardiolipin [aCL] IgG and IgM; anti-ß(2)glycoprotein I [anti-ß(2)GPI] antibodies IgG and IgM; and/or a lupus anticoagulant [LAC]), when at least one of the two major clinical manifestations (thrombosis or pregnancy losses) are present. Although, efforts of standardization for these 'criteria' aPL tests have been conducted over the last 27 years, reports of inconsistencies, inter-assay and inter-laboratory variation in the results of aCL, LAC, and anti-ß(2)GPI, and problems with the interpretation and the clinical value of the tests still exist, which affect the consistency of the diagnosis of APS. A Task Force of scientists and pioneers in the field from different countries, subdivided in three working groups, discussed and analyzed critical questions related to 'criteria' aPL tests in an evidence-based manner, during the 13(th) International Congress on Antiphospholipid Antibodies (APLA 2010, April 13-16, 2010, Galveston, TX). These included: review of the standardization and the need for international consensus protocol for aCL and anti-ß(2)GPI tests; the use of monoclonal and/or polyclonal standards in the calibration curve of those tests; and the need for establishment of international units of measurement for anti-ß(2)GPI tests. The group also reviewed the recently updated guidelines for LAC testing, and analyzed and discussed the possibility of stratification of 'criteria' aPL tests as risk factors for APS, as well as the clinical value of single positive vs. multiple aPL positivity. The group members presented, discussed, analyzed data, updated and re-defined those critical questions at a preconference workshop that was open to congress attendees. This report summarizes the findings, conclusions, and recommendations of this Task Force.

Fibrinogen-coated albumin microcapsules reduce bleeding in severely thrombocytopenic rabbits.

Severe thrombocytopenia frequently occurs in patients receiving chemotherapy and in patients with autoimmune disorders. Thrombocytopenia is associated with bleeding, which may be serious and life threatening. Current treatment strategies for thrombocytopenia may require transfusion of allogeneic platelets, which is associated with serious drawbacks. These include the occurrence of anti-platelet antibodies, which may result in refractoriness to further platelet transfusions, and the potential risk of transfer of blood-borne diseases. Therefore, we have recently developed a platelet substitute product (Synthocytes), which is composed of human albumin microcapsules with fibrinogen immobilized on their surface. Here we show that the intravenous administration of these microcapsules not only corrects the prolonged bleeding time in rabbits rendered thrombocytopenic either by anti-platelet antibodies or by chemotherapy, but also reduces bleeding from surgical wounds inflicted in the abdominal skin and musculature. No potential systemic prothrombotic effect of the microcapsules was observed in a model of rabbit venous thrombosis. As for the mechanism of action, experiments with normal and thrombocytopenic human blood in an endothelial cell matrix-coated perfusion chamber demonstrated an interaction between the fibrinogen-coated albumin microcapsules and native platelets. It was shown that the fibrinogen-coated albumin microcapsules could facilitate platelet adhesion to endothelial cell matrix and correct the impaired formation of platelet aggregates in relatively platelet-poor blood. This study indicates that fibrinogen-coated albumin microcapsules can act to improve primary hemostasis under thrombocytopenic conditions and may eventually be a promising agent for prophylaxis and treatment of bleeding in patients with severe thrombocytopenia.

Towards evidence-based treatment of thrombotic antiphospholipid syndrome.

Thrombosis in the presence of persistently positive tests for antiphospholipid antibodies is termed thrombotic antiphospholipid syndrome (APS). At present, 'standard' secondary thromboprophylaxis in thrombotic APS is treatment with moderate intensity oral anticoagulants for life after a first venous thrombosis and with high intensity oral anticoagulation after non-embolic ischaemic stroke. These recommendations differ from those applied in the general population, where a restricted period of anticoagulation is common practice after venous thrombosis and antiplatelet drugs are the first choice after ischaemic stroke. From an extensive literature review we conclude that the available data are insufficient to apply a different strategy for secondary thromboprophylaxis in patients with thrombotic APS than the one that holds for the general population.

The role of LRP8 (ApoER2') in the pathophysiology of the antiphospholipid syndrome.

One of the greatest enigmas in thrombosis research is the observation that one can diagnose a person with a thrombotic risk with a prolongation of the clotting time. Our textbooks have taught us that prolongation of clotting correlates with a tendency to bleed. To confuse our textbook knowledge further, the same patients often have a prolonged bleeding time, a diagnostic test to detect a dysfunction in primary haemostasis. In this paper we critically review the literature that tries to explain the contradiction that exists between in-vitro diagnostic tests and the observed clinical manifestations and discuss our current opinion on how antiphospholipid antibodies can disturb the haemostatic balance.

Antiphospholipid syndrome. Current insights into laboratory diagnosis and pathophysiology.

The antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL) in the plasma of patients with vascular thrombosis, recurrent complications of pregnancy, or both (1, 2). The presence of aPL in plasma of patients can be detected with either a prolongation of phospholipid dependent coagulation tests (lupus anticoagulant, LAC), or with solid phase immune assays against the protein beta2-glycoprotein I (beta2-GPI) or the phospholipid cardiolipin (anti-beta2-GPI antibody ELISA and anti-cardiolipin antibody ELISA, respectively) (3). For a long time there was a lot of confusion on who had the syndrome and who not. To solve this dispute, an international consensus meeting was organized in Sapporo in 1999 to formulate classification criteria for patients with the antiphospholipid syndrome (4). These criteria have been updated in 2004 at another international consensus meeting in Sydney (5). The classification criteria were defined for scientific purposes and were aimed to be used as inclusion criteria in patient related studies. They were specifically not defined for diagnostic purposes. However, current practice is that these criteria are used as a diagnostic tool. This is very unfortunate because the specificity of the different aPL assays to detect the clinical manifestations that characterize APS are disputable. One of the aims of defining the criteria was to initiate studies to determine the value of the different anti-phospholipid antibody assays to serve as biomarker for the risk of thrombosis and pregnancy morbidity. The recent progress made on this important topic will be discussed.

Morbidity and mortality in the antiphospholipid syndrome during a 5-year period: a multicentre prospective study of 1000 patients.

OBJECTIVES: To identify the main causes of morbidity and mortality in patients with antiphospholipid syndrome (APS) during a 5-year period and to determine clinical and immunological parameters with prognostic significance. METHODS: The clinical and immunological features of a cohort of 1000 patients with APS from 13 European countries who had been followed up from 1999 to 2004 were analysed. RESULTS: 200 (20%) patients developed APS-related manifestations during the 5-year study period. Recurrent thrombotic events appeared in 166 (16.6%) patients and the most common were strokes (2.4% of the total cohort), transient ischaemic attacks (2.3%), deep vein thromboses (2.1%) and pulmonary embolism (2.1%). When the thrombotic events occurred, 90 patients were receiving oral anticoagulants and 49 were using aspirin. 31/420 (7.4%) patients receiving oral anticoagulants presented with haemorrhage. 3/121 (2.5%) women with only obstetric APS manifestations at the start of the study developed a new thrombotic event. A total of 77 women (9.4% of the female patients) had one or more pregnancies and 63 (81.8% of pregnant patients) had one or more live births. The most common fetal complications were early pregnancy loss (17.1% of pregnancies) and premature birth (35% of live births). 53 (5.3% of the total cohort) patients died. The most common causes of death were bacterial infection (21% of deaths), myocardial infarction (19%) and stroke (13%). No clinical or immunological predictor of thrombotic events, pregnancy morbidity or mortality was detected. CONCLUSION: Patients with APS still develop significant morbidity and mortality despite current treatment (oral anticoagulants or antiaggregants, or both).

Elevated plasma surfactant protein D (SP-D) levels and a direct correlation with anti-severe acute respiratory syndrome coronavirus-specific IgG antibody in SARS patients.

Pulmonary SP-D is a defence lectin promoting clearance of viral infections. SP-D is recognized to bind the S protein of SARS-CoV and enhance phagocytosis. Moreover, systemic SP-D is widely used as a biomarker of alveolar integrity. We investigated the relation between plasma SP-D, SARS-type pneumonia and the SARS-specific IgG response. Sixteen patients with SARS, 19 patients with community-acquired pneumonia (CAP) (Streptococcus pneumonia) and 16 healthy control subjects were enrolled in the study. Plasma SP-D and anti-SARS-CoV N protein IgG were measured using ELISA. SP-D was significantly elevated in SARS-type pneumonia [median (95% CI), 453 (379-963) ng/ml versus controls 218 (160-362) ng/ml, P < 0.05] like in patients with CAP. SP-D significantly correlated with anti-SARS-CoV N protein IgG (r(2) = 0.5995, P = 0.02). The possible re-emergence of SARS or SARS-like infections suggests a need for minimal traumatic techniques for following the alveolar compartment, e.g. during testing of antivirals. We suggest that monitoring systemic SP-D may be useful in monitoring the alveolar integrity in SARS-type pneumonia. The significant correlation between plasma SP-D and anti-SARS-CoV-specific antibodies support the role for SP-D in interlinking innate and adaptive immune pathways.

Perturbation of human endothelial cells by thrombin or PMA changes the reactivity of their extracellular matrix towards platelets.

In this study we have examined the influence of perturbation of endothelial cells on the amounts of fibronectin and von Willebrand factor in their extracellular matrix and the consequences of a changed composition of the matrix on platelet adhesion. For this purpose, we have used an in vitro perfusion system with which we can investigate the interactions of platelets in flowing blood with cultured endothelial cells and their extracellular matrix (Sakariassen, K. S., P. A. M. M. Aarts, P. G. de Groot, W. P. M. Houdgk, and J. J. Sixma, 1983, J. Lab. Clin Med. 102:522-535). Treatment of endothelial cells with 0.1-1.0 U/ml thrombin for 2 h increased the reactivity of the extracellular matrix, isolated after the thrombin treatment, towards platelets by approximately 50%. The increased reactivity did not depend on de novo protein synthesis but was inhibited by 3-deazaadenosine, an inhibitor of phospholipid methylation, which also inhibits the stimulus-induced instantaneous release of von Willebrand factor from endothelial cells. However, no changes in the amounts of von Willebrand factor and fibronectin in the matrix were detected. Thrombin may change the organization of the matrix proteins, not the composition. When endothelial cells were perturbed with the phorbol ester PMA or thrombin for 3 d, the adhesion of platelets to the extracellular matrix of treated cells was strongly impaired. This impairment coincided with a decrease in the amounts of von Willebrand factor and fibronectin present in the matrix. These results indicate that, after perturbation, endothelial cells regulate the composition of their matrix, and that this regulation has consequences for the adhesion of platelets.

High prevalence of reduced thrombin generation and/or decreased platelet response in women with unexplained heavy menstrual bleeding.

INTRODUCTION: Heavy menstrual bleeding (HMB) is a condition that affects 20%-30% of women of reproductive age. HMB has a multifactorial pathophysiology, which is incompletely understood. HMB symptoms are very common in patients with established haemostasis defects, likewise, women with heavy menstrual bleeding have a higher prevalence of impaired Von Willebrand factor (VWF) levels and function, thrombocytopenia, impaired platelet function and impaired coagulation. The aim of this study was to quantify the prevalence of impaired platelet function, impaired coagulation and reduced VWF activity in patients with HMB. METHODS: We have used thrombin generation (TG), a flow cytometry-based platelet function test and a flow cytometry-based VWF function test to study haemostasis in 58 women (median age: 48.4 years, range 40-60 years) with HMB. In addition, we determined VWF antigen levels and VWF ristocetin co-factor activity in platelet-poor plasma. Reference ranges of platelet function were measured in whole blood of 123 healthy volunteers, while reference ranges of TG were determined in platelet-poor plasma (PPP) of 126 healthy volunteers. RESULTS: Fourteen (24%) patients with HMB had impaired platelet function and 17 (29.3%) patients had impaired coagulation. Five patients (8.6%) had both impaired platelet function and impaired coagulation. Only 2 (3.4%) patients had an impaired VWF function or levels; one of them was in combination with impaired coagulation. CONCLUSION: Our approach in women with HMB using a high precision platelet function test in combination with thrombin generation showed impaired coagulation or impaired platelet function in more than 40% of the patients.

Absence of ligands bound to glycoprotein IIB-IIIA on the exposed surface of a thrombus may limit thrombus growth in flowing blood.

We examined the distribution of glycoprotein IIb-IIIa (GPIIb-IIIa) and its ligands fibrinogen and von Willebrand factor (vWf) on platelets which had adhered under flow conditions. Immunoelectron microscopy was performed on whole mounts and frozen thin sections of adhering platelets. GPIIb-IIIa was homogeneously distributed on dendritic platelets and on interplatelet membranes of formed thrombi. Fibrinogen and vWf were predominantly associated with interplatelet membranes and membranes facing the substrate. On whole mounts, vWf appeared in clumps and linear arrays, representing the tangled or extended forms of the multimeric molecule. From semiquantitative analysis, it appeared that fibrinogen and vWf were, respectively, nine- and fourfold higher on interplatelet membranes than on surface membranes facing the blood stream, while GPIIb-IIIa was evenly distributed over all platelet plasma membranes. Ligand-induced binding sites (LIBS) of GPIIb-IIIa, as measured with conformation specific monoclonal antibodies RUU 2.41 and LIBS-1, were present on the surface of adhered platelets and thrombi. A redistribution of LIBS-positive forms of GPIIb-IIIa towards interplatelet membranes was not observed. Our data support the hypothesis that, under flow conditions, ligands have first bound to activated GPIIb-IIIa but this binding is reversed on the upper surface of adhering platelets. This relative absence of ligands on the exposed surface of thrombi may play a role in limiting their size.

Evidence that the primary binding site of von Willebrand factor that mediates platelet adhesion on subendothelium is not collagen.

We have studied the binding of von Willebrand factor to extracellular matrices of endothelial cells and to the vessel wall of human umbilical arteries in relation to its function in supporting platelet adhesion. CLB-RAg 201, an MAb against von Willebrand factor, completely inhibits the binding of von Willebrand factor to collagen type I and type III. CLB-RAg 201 does not inhibit the binding of 125I-von Willebrand factor to extracellular matrices of endothelial cells, to smooth muscle cells, or to the subendothelium. CLB-RAg 201 partly inhibits platelet adhesion to these surfaces, but this directly affects the interaction between von Willebrand factor and platelets and is not due to inhibition of binding of von Willebrand factor to these surfaces. Another MAb, CLB-RAg 38, does not inhibit the binding of von Willebrand factor to collagen. CLB-RAg 38 completely inhibits the binding of von Willebrand factor to extracellular matrices. CLB-RAg 38 inhibits platelet adhesion to cellular matrices completely insofar as it is dependent on plasma von Willebrand factor. CLB-RAg 38 does not inhibit the total binding of von Willebrand factor to subendothelium, as there are too many different binding sites, but it completely inhibits the functional binding sites for von Willebrand factor that support platelet adhesion. The epitopes for CLB-RAg 38 and 201 on the von Willebrand factor molecule are located on different fragments of the molecule. These results indicate that von Willebrand factor binds to subendothelium and matrices of cultured cells by a mechanism that is different from that by which it binds to collagen.

Thrombospondin-platelet interactions. Role of divalent cations, wall shear rate, and platelet membrane glycoproteins.

The role of thrombospondin, a multifunctional matrix glycoprotein, in platelet adhesion is controversial: both adhesive and antiadhesive properties have been attributed to this molecule. Because shear flow has a significant influence on platelet adhesion, we have assessed thrombospondin-platelet interactions both under static and flow conditions. The capacity of thrombospondin to support platelet adhesion depended upon its conformation. In a Ca(2+)-depleted conformation, such as in citrated plasma, thrombospondin was nonadhesive or antiadhesive as it inhibited platelet adhesion to fibrinogen, fibronectin, laminin, and von Willebrand factor by 30-70%. In a Ca(2+)-replete conformation, however, thrombospondin effectively supported platelet adhesion. Shear rate influenced this adhesion; percent surface coverage on thrombospondin increased from 5.4 +/- 0.3 at 0 s-1 to 41.5 +/- 6.7 at 1,600 s-1. In contrast to the extensive platelet spreading observed on fibronectin at all shear rates, platelet spreading on thrombospondin occurred only sporadically and at high shear rates. GPIa-IIa, GPIIb-IIIa, GPIV, and the vitronectin receptor, which are all proposed platelet receptors for thrombospondin, were not solely responsible for platelet adhesion to thrombospondin. These results suggest that thrombospondin may play a dual role in adhesive processes in vivo: (a) it may function in conjunction with other adhesive proteins to maintain optimal platelet adhesion at various shear rates; and (b) it may serve as a modulator of cellular adhesive functions under specific microenvironmental conditions.

Increased platelet deposition on atherosclerotic coronary arteries.

A ruptured atherosclerotic plaque leads to exposure of deeper layers of the plaque to flowing blood and subsequently to thrombus formation. In contrast to the wealth of data on the occurrence of thrombi, little is known about the reasons why an atherosclerotic plaque is thrombogenic. One of the reasons is the relative inaccessibility of the atherosclerotic plaque. We have circumvented this problem by using 6-microns cryostat cross sections of human coronary arteries. These sections were mounted on coverslips that were exposed to flowing blood in a rectangular perfusion chamber. In normal-appearing arteries, platelet deposition was seen on the luminal side of the intima and on the adventitia. In atherosclerotic arteries, strongly increased platelet deposition was seen on the connective tissue of specific parts of the atherosclerotic plaque. The central lipid core of an advanced plaque was not reactive towards platelets. The results indicate that the atherosclerotic plaque by itself is more thrombogenic than the normal vessel wall. To study the cause of the increased thrombus formation on the atherosclerotic plaque, perfusion studies were combined with immunohistochemical studies. Immunohistochemical studies of adhesive proteins showed enrichment of collagen types I, III, V, and VI, vitronectin, fibronectin, fibrinogen/fibrin, and thrombospondin in the atherosclerotic plaque. Laminin and collagen type IV were not enriched. von Willebrand Factor (vWF) was not present in the plaque. The pattern of increased platelet deposition in serial cross sections corresponded best with areas in which collagen types I and III were enriched, but there were also areas in the plaque where both collagens were enriched but no increased reactivity was seen. Inhibition of platelet adhesion with a large range of antibodies or specific inhibitors showed that vWF from plasma and collagen types I and/or III in the plaque were involved. Fibronectin from plasma and fibronectin, fibrinogen, laminin, and thrombospondin in the vessel wall had no effect on platelet adhesion. We conclude that the increased thrombogenicity of atherosclerotic lesions is due to changes in quantity and nature of collagen types I and/or III.

Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen.

We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35.