Jelle Adelmeijer

Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity.

Cirrhosis of the liver is frequently accompanied by complex alterations in the hemostatic system, resulting in a bleeding tendency. Although many hemostatic changes in liver disease promote bleeding, compensatory mechanisms also are found, including high levels of the platelet adhesive protein von Willebrand Factor (VWF). However, conflicting reports on the functional properties of VWF in cirrhosis have appeared in literature. We have measured a panel of VWF parameters in plasma from patients with cirrhosis of varying severity and causes. Furthermore, we assessed the contribution of VWF to platelet adhesion, by measuring the ability of plasma from patients with cirrhosis to support adhesion of normal or patient platelets under flow conditions. VWF antigen levels were strongly increased in patients with cirrhosis. In contrast, the relative collagen binding activity, as well as the relative ristocetin cofactor activity, was significantly lower in patients as compared with controls, indicating loss of function. Accordingly, patients had a reduced fraction of high‐molecular‐weight VWF multimers. Both strongly elevated and reduced activity and antigen levels of the VWF cleaving protease ADAMTS13 were found in individual patients. Adhesion of either normal or patient platelets to a collagen surface was substantially increased when these platelets were resuspended in plasma of patients with cirrhosis, as compared with control plasma. In conclusion, highly elevated levels of VWF in patients with cirrhosis contribute to the induction of primary hemostasis despite reduced functional properties of the molecule. This phenomenon might compensate for defects in platelet number and function in patients with cirrhosis. (HEPATOLOGY 2006;44:53–61.)

Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1

Collagens are the most abundant proteins in the human body, important in maintenance of tissue structure and hemostasis. Here we report that collagens are high affinity ligands for the broadly expressed inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). The interaction is dependent on the conserved Gly-Pro-Hyp collagen repeats. Antibody cross-linking of LAIR-1 is known to inhibit immune cell function in vitro. We now show that collagens are functional ligands for LAIR-1 and directly inhibit immune cell activation in vitro. Thus far, all documented ligands for immune inhibitory receptors are membrane molecules, implying a regulatory role in cell–cell interaction. Our data reveal a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens.

Platelet adhesion to dimeric β2‐glycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein Ibα and apolipoprotein E receptor 2′

Summary.  Background: The major antigen implicated in the antiphospholipid syndrome is beta2‐glycoprotein I (β2GPI). Dimerized β2GPI binds to apolipoprotein E receptor 2′ (apoER2′) on platelets and increases platelet adhesion to collagen under conditions of flow. Aim: To investigate whether the interaction between dimerized β2GPI and platelets is sufficiently strong to resist shear stresses. Methods: We studied the interaction of platelets with immobilized dimerized β2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. Results: We found that dimerized β2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized β2GPI was completely inhibited by the addition of soluble forms of both apoER2′ and GPIbα, and the addition of receptor‐associated protein and the removal of GPIbα from the platelet surface. GPIbα co‐precipitated with apoER2′, suggesting the presence of complexes between GPIbα and apoER2′ on platelet membranes. The interaction between GPIbα and dimeric β2GPI was of intermediate affinity (Kd = 180 nm) and Zn2+, but not Ca2+‐dependent. Deletion of domain V from dimeric β2GPI strongly reduced its binding to both GPIbα and apoER2′. Antibodies that inhibit the binding of thrombin to GPIbα inhibited platelet adhesion to dimeric β2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIbα had no effect. Dimeric β2GPI showed reduced binding to low‐sulfated GPIbα compared to the fully sulfated form. Conclusion: We show that platelets adhere to dimeric β2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIbα and apoER2′. These receptors are present in a complex on the platelet surface.

Platelet Activation by Oxidized Low Density Lipoprotein Is Mediated by Cd36 and Scavenger Receptor-A

Objective—The interaction of platelets with low density lipoprotein (LDL) contributes to the development of cardiovascular disease. Platelets are activated by native LDL (nLDL) through apoE Receptor 2′ (apoER2′)-mediated signaling to p38MAPK and by oxidized LDL (oxLDL) through lysophosphatidic acid (LPA) signaling to Rho A and Ca2+. Here we report a new mechanism for platelet activation by oxLDL. Methods and Results—Oxidation of nLDL increases p38MAPK activation through a mechanism that is (1) independent of LPA, and (2) unlike nLDL-signaling not desensitized by prolonged platelet-LDL contact or inhibited by receptor-associated protein or chondroitinase ABC. Antibodies against scavenger receptors CD36 and SR-A alone fail to block p38MAPK activation by oxLDL but combined blockade inhibits p38MAPK by >40% and platelet adhesion to fibrinogen under flow by >60%. Mouse platelets deficient in either CD36 or SR-A show normal p38MAPK activation by oxLDL but combined deficiency of CD36 and SR-A disrupts oxLDL-induced activation of p38MAPK by >70%. Conclusion—These findings reveal a novel platelet-activating pathway stimulated by oxLDL that is initiated by the combined action of CD36 and SR-A.

An unbalance between von Willebrand factor and ADAMTS13 in acute liver failure: implications for hemostasis and clinical outcome.

Emerging evidence supports the concept of a rebalanced hemostatic state in liver disease as a result of a commensurate decline in prohemostatic and antihemostatic drivers. In the present study, we assessed levels and functionality of the platelet‐adhesive protein von Willebrand factor (VWF) and its cleaving protease ADAMTS13 in the plasma of patients with acute liver injury and acute liver failure (ALI/ALF). Furthermore, we explored possible associations between VWF, ADAMTS13, and disease outcome. We analyzed the plasma of 50 patients taken on the day of admission for ALI/ALF. The plasma of 40 healthy volunteers served as controls. VWF antigen levels were highly elevated in patients with ALI/ALF. In contrast, the collagen‐binding activity and the ratio of the VWF ristocetin cofactor activity and VWF antigen was significantly decreased when compared with healthy controls. Also, the proportion of high molecular weight VWF multimers was reduced, despite severely decreased ADAMTS13 levels. In spite of these functional defects, platelet adhesion and aggregation were better supported by plasma of patients with ALI/ALF when compared with control plasma. Low ADAMTS13 activity, but not high VWF antigen, was associated with poor outcome in patients with ALI/ALF as evidenced by higher grades of encephalopathy, higher transplantation rates, and lower survival. VWF or ADAMTS13 levels were not associated with bleeding or thrombotic complications. Conclusion: Highly elevated levels of VWF in plasma of patients with ALI/ALF support platelet adhesion, despite a relative loss of function of the molecule. Furthermore, low ADAMTS13 activity is associated with progressive liver failure in the patient cohort, which might be attributed to platelet‐induced microthrombus formation in the diseased liver resulting from a substantially unbalanced VWF/ADAMTS13 ratio. (Hepatology 2013;58:752–761)

The glycoprotein Ib-IX-V complex contributes to tissue factor-independent thrombin generation by recombinant factor VIIa on the activated platelet surface

Several lines of evidence suggest that recombinant factor VIIa (rFVIIa) is able to activate factor X on an activated platelet, in a tissue factor-independent manner. We hypothesized that, besides the anionic surface, a receptor on the activated platelet surface is involved in this process. Here, we showed that, in an ELISA setup, a purified extracellular fragment of GPIbalpha bound to immobilized rFVIIa. Surface plasmon resonance established a affinity constant (K(d)) of approximately 20 nM for this interaction. In addition, CHO cells transfected with the GPIb-IX-V complex could adhere to immobilized rFVIIa, whereas wild-type CHO cells could not. Furthermore, platelets sti-mulated with a combination of collagen and thrombin adhered to immobilized rFVIIa under static conditions. Platelet adhesion was inhibited by treatment with O-sialoglycoprotein endopeptidase, which specifically cleaves GPIbalpha from the platelet surface. In addition, rFVIIa-mediated thrombin generation on the activated platelet surface was inhibited by cleaving GPIbalpha from its surface. In summary, 3 lines of evidence showed that rFVIIa interacts with the GPIb-IX-V complex, and this interaction enhanced tissue factor-independent thrombin generation mediated by rFVIIa on the activated platelet surface. The rFVIIa-GPIbalpha interaction could contribute to cessation of bleeding after administration of rFVIIa to patients with bleeding disorders.

Development of a Severe von Willebrand Factor/ADAMTS13 Dysbalance During Orthotopic Liver Transplantation

Patients with liver disease show profound changes in their hemostatic system, which may further change during liver transplantation. We previously demonstrated that highly elevated levels of the platelet adhesive protein von Willebrand factor (VWF) in patients with cirrhosis lead to an increased VWF‐dependent platelet deposition under flow as compared to healthy controls. In this study we examined VWF parameters during the course of liver transplantation. We collected serial plasma samples from 20 patients undergoing liver transplantation in which we determined plasma levels of VWF and the VWF‐cleaving protease ADAMTS13. Furthermore, we performed functional tests of VWF‐dependent platelet adhesion. We found persistently elevated levels of VWF during and after liver transplantation. The capacity of VWF to interact with platelets normalized during the course of transplantation, and flow‐mediated VWF‐dependent platelet adhesion remained at levels far exceeding those observed in healthy individuals during and after transplantation. Plasma levels of ADAMTS13 dropped during transplantation, and in four patients levels below 10% of normal were observed after reperfusion. We observed the development of a hyperreactive primary hemostatic system, as evidenced by high levels of fully functional VWF and a temporary ADAMTS13 deficiency, during liver transplantation, and speculate that these changes contribute to postoperative thrombotic complications.

Interlaboratory variability in assessment of the model of end‐stage liver disease score

Background: The model of end‐stage liver disease (MELD) score is nowadays widely used to prioritize patients for liver transplantation.

Intact thrombin generation and decreased fibrinolytic capacity in patients with acute liver injury or acute liver failure

Summary.  Background:  It has been well established that hemostatic potential in patients with chronic liver disease is in a rebalanced status due to a concomitant decrease in pro‐ and antihemostatic drivers. The hemostatic changes in patients with acute liver injury/failure (ALI/ALF) are similar but not identical to the changes in patients with chronic liver disease and have not been studied in great detail.

No evidence for an intrinsic platelet defect in patients with liver cirrhosis – studies under flow conditions

See also Reverter JC. Abnormal hemostasis tests and bleeding in chronic liver disease: are they related? Yes. J Thromb Haemost 2006; 4: 717–20; Mannucci PM. Abnormal hemostasis tests and bleeding in chronic liver disease: are they related? No. J Thromb Haemost 2006; 4: 721–3; Lisman T, Caldwell SH, Leebeek FWG, Porte RJ; Lisman T, Porte RJ, Leebeek FWG, Caldwell SH; Lisman T, Caldwell SH, Porte RJ, Leebeek FWG; Tripodi A; Violi F; Matsushita T, Saito H; Laffi G, Marra F, Tarquini R, Abbate R. Hemostasis in chronic liver disease. This issue, pp 2059–69.