J. A. Van Mourik

Association of idiopathic venous thromboembolism with single point-mutation at Arg506 of factor V

Abnormal coagulation factor V may underlie the thrombotic events associated with resistance to activated protein C (APC). We analysed 27 consecutive patients with documented idiopathic (recurrent) thromboembolism for the occurrence of point mutations within the APC sensitive regions of blood coagulation factor V. In 10 patients we observed a single basepair mutation resulting in a substitution of Arg506 to Gln. This mutation was significantly linked to in-vitro resistance to APC in these subjects. This mutation at Arg506 of factor V may form the molecular basis for the thrombotic events associated with APC resistance.

Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family.

A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full‐length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full‐length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk‐ cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti‐plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk‐ cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre‐PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine‐linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1‐proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)

The Light Chain of Factor VIII Comprises a Binding Site for Low Density Lipoprotein Receptor-related Protein

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K d ≈ 60 nm). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC50 ≈ 1 nm). In addition, a 12-fold molar excess of the physiological carrier of FVIII,i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of125I-labeled FVIII by LRP-expressing cells (≈ 8 fmol/105 cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k on≈ 5 × 104 m −1s−1; k off ≈ 2.5 × 10−3 s−1; K d ≈ 50 nm). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on thein vivo survival of FVIII.

Media conditioned by cultured human vascular endothelial cells inhibit the growth of vascular smooth muscle cells

Abstract To determine if human vascular endothelium influences the growth of smooth muscle cells (SMC), we have investigated the effect of media conditioned by cultured human vascular endothelial cells on smooth muscle cell proliferation. Cell growth was measured by [ 3 H]thymidine incorporation in endothelial and smooth muscle cells. The conditioned medium of confluent endothelial cells contains factors that inhibit the growth of actively dividing endothelial and smooth muscle cells. Media conditioned by proliferating endothelial cells or confluent smooth muscle cells were inactive. The generation of the inhibitory activity was time-dependent, not correlated with cell lysis and was demonstrated in both serum-free and serum-containing medium. Protein synthesis was involved in the generation of the inhibitory activity. Upon gel filtration, the inhibitory activity was associated with a substance having a molecular weight (MW) of >10 5 . Treatment with trypsin or alpha chymotrypsin did not abolish the activity. Furthermore, the activity remained stable after incubation for 30 min at 56 °C, whereas only 50% of the activity remained present after heating for 2 min at 100 °C. It is speculated that this inhibitory activity plays an important role in the regulation of smooth muscle cell proliferation.

Cell cycle-dependent inhibition of human vascular smooth muscle cell proliferation by prostaglandin E1

We examined the influence of prostaglandins on the initiation of proliferation of growth-arrested human adult aortic and fetal smooth muscle cells. Prostaglandins of the E series (25 nM) exerted a significant (p less than or equal to 0.05) inhibitory effect on DNA synthesis. Inhibition was observed when PGE1 was added in the G1 phase of the cell cycle. PGE1 had no effect when added once DNA synthesis had started. Thus prostaglandins of the E series may inhibit the responsiveness of smooth muscle cells to the mitogenic action of critical growth factors, such as PGDF. This inhibitory response is cell-cycle dependent. Once smooth muscle cells have entered S phase, PGE1 is no longer effective. Our data also suggest that cAMP is involved in the PGE1-induced growth inhibition, since concomitant with PGE1 addition, cAMP levels rose rapidly; addition of the cAMP analogue db-cAMP resulted in a cell-cycle-dependent inhibition pattern comparable to that observed with PGE1.

ADAMTS-13, von Willebrand factor and related parameters in severe sepsis and septic shock

Background: Insufficient control of von Willebrand factor (VWF) multimer size as a result of severely deficient ADAMTS‐13 activity results in thrombotic thrombocytopenic purpura associated with microvascluar thrombosis and platelet consumption, features not seldom seen in severe sepsis and septic shock. Methods: ADAMTS‐13 activity and VWF parameters of 40 patients with severe sepsis or septic shock were compared with those of 40 healthy controls of the same age and gender and correlated with clinical findings and sepsis outcome. Results: ADAMTS‐13 activity was significantly lower in patients than in healthy controls [median 60% (range 27–160%) vs. 110% (range 63–200%); P < 0.001]. VWF parameters behaved reciprocally and both VWF ristocetin cofactor activity (RCo) and VWF antigen (VWF:Ag) were significantly (P < 0.001) higher in patients compared with controls. Neither ADAMTS‐13 activity nor VWF parameters correlated with disease severity, organ dysfunction or outcome. However, a contribution of acute endothelial dysfunction to renal impairment in sepsis is suggested by the significantly higher VWF propeptide and soluble thrombomodulin levels in patients with increased creatinine values as well as by their strong positive correlations (creatinine and VWF propeptide rs = 0.484, P < 0.001; creatinine and soluble thrombomodulin rs = 0.596, P < 0.001). Conclusions: VWF parameters are reciprocally correlated with ADAMTS‐13 activity in severe sepsis and septic shock but have no prognostic value regarding outcome.

Biogenesis of von Willebrand factor-containing organelles in heterologous transfected CV-1 cells.

Von Willebrand factor (vWF) is a multimeric protein involved in the adhesion of platelets to an injured vessel wall. vWF is synthesized by the endothelial cell and the megakaryocyte as a precursor protein (pro‐vWF) that consists of four repeated domains, denoted D1‐D2‐D′‐D3‐A1‐A2‐A3‐D4‐B1‐B2‐B3‐C1‐C2. Previously, we have defined the domains on the pro‐vWF molecule involved in dimerization as well as the domains involved in multimer assembly of vWF dimers. In the endothelial cell, part of the vWF multimers is stored in specialized organelles, the Weibel‐Palade bodies. By using immunoelectron microscopy, we demonstrate that upon expression of full‐length vWF cDNA, vWF‐containing organelles are encountered in monkey kidney CV‐1 cells that are morphologically similar to the endothelial‐specific Weibel‐Palade bodies. Expression in CV‐1 cells of a series of vWF cDNA deletion mutants, lacking one or more domains, revealed that only those vWF mutant proteins that are able to assemble into multimers are encountered in dense‐cored vesicles. Our data show that this process is independent of a particular domain on vWF and indicate that a ‘condensed’, multimeric vWF is required for targeting to the Weibel‐Palade body.

Cigarette smoke impairs endothelial cell prostacyclin production.

Production of prostacyclin by endothelial cells is considered to be important in rendering the vessel wall nonthrombogenic. Cigarette smoking is an important risk factor in the pathogenesis of atherosclerosis. Here we show that the incubation of cultured human endothelial cells with a cigarette smoke condensate impaired the basal prostacyclin release. Also, the enhanced release of prostacyclin provoked by phorbol myristate acetate was inhibited by cigarette smoke condensate. Furthermore, cigarette smoke condensate impaired the thrombininduced prostacyclin production. The production of prostacyclin from exogenous arachidonate was not affected by cigarette smoke condensate, indicating that cigarette smoke condensate constituents exert their inhibitory properties on the level of arachidonate mobilization from cellular phospholipids, rather than on cyclooxygenase or prostaglandin synthetase. The effects noted for cigarette smoke condensate could not be attributed to the cigarette smoke constituents nicotine and cadmium. While inhibiting the endothelial cell prostacyclin production significantly, cigarette smoke condensate did not cause cell death or impairment of secretory function, as measured by the release of von Willebrand factor. This in vitro study shows that impairment of an endothelial cell function is related to a risk factor for atherosclerosis.

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.

von Willebrand factor and its propeptide: the influence of secretion and clearance on protein levels and the risk of venous thrombosis.

Summary.  Background and objectives: Elevated levels of factor (F)VIII are associated with an increased risk of thrombosis. FVIII levels are determined mainly by von Willebrand factor (VWF). We have investigated the contribution of secretion and clearance rates to the elevated VWF antigen (VWF:Ag) and to the risk of thrombosis. VWF is secreted in equimolar amounts with its propeptide, which has a shorter half‐life. VWF propeptide can be used as a measure of VWF secretion and allows estimation of the VWF half‐life. Methods and results: We have measured VWF propeptide, VWF:Ag, FVIII:Ag and FVIII activity (FVIII:C) in the Leiden Thrombophilia Study. In controls, high VWF propeptide was associated with high VWF:Ag, FVIII:Ag and FVIII:C. In contrast to mature VWF:Ag, VWF propeptide was not influenced by blood groups. Using an ELISA‐based assay we have shown that VWF propeptide lacks ABO antigens. Levels were higher in men and increased with age. A long VWF half‐life was also associated with high VWF:Ag, FVIII:Ag and FVIII:C. The VWF half‐life was influenced by blood group (10 h in O vs. 12 h in non‐O individuals), but not by sex, and only slightly by age. VWF propeptide was higher in thrombosis patients than in controls. The VWF half‐life was similar in patients and controls (11.4 and 11.1 h, respectively). Conclusions: Both secretion and clearance rates are important determinants of VWF and FVIII levels. However, mainly high VWF and FVIII levels caused by increased secretion seem to be associated with thrombosis. ABO blood group influences the clearance rates of VWF rather than VWF secretion rates.