Endothelial Cell-derived Von Willebrand Factor Research Articles

Indications for a protective function of beta2‐glycoprotein I in thrombotic thrombocytopenic purpura

It has been shown that β(2) -glycoprotein I (β(2) GPI) interacts with von Willebrand factor (VWF) in a glycoprotein (GP)Ib binding state. Given the presence of active VWF multimers in thrombotic thrombocytopenic purpura (TTP), we speculated that β(2) GPI might play a role in TTP. We found that β(2) GPI plasma levels were significantly lower in acute and remission TTP patients than in normal controls, showing a direct correlation with ADAMTS 13 levels and an inverse correlation with the extent of VWF activation. In vitro flow experiments demonstrated that β(2) GPI can block platelet adhesion to endothelial cell-derived VWF strings. We confirmed the direct binding of β(2) GPI to VWF by surface plasmon resonance, and determined that domain I of β(2) GPI is the binding site of VWF A1 domain. Adhesion of β(2) GPI to erythrocytes and platelets was increased in the presence of active VWF, indicating that β(2) GPI may be cleared from the circulation during TTP episodes together with blood cells. Our findings suggest that β(2) GPI may protect from the effects of hyper-functional VWF by inhibiting its interaction with platelets.

The Role of VWF In BBB Permeability Associated with Hypoxia/Reoxygenation

AbstractAbstract 2102Aberrant blood brain barrier (BBB) permeability is a hallmark pathology in many diseases of the central nervous system (CNS) including hypoxia, epilepsy, multiple sclerosis and ischemic stroke. Generalized hypoxia is a pathological condition in which the body as a whole is deprived of adequate oxygen supply. Hypoxia occurs in healthy people when they ascend to high altitudes, where it can cause altitude sickness, often manifested by headache, leading to potentially fatal complications such as high altitude cerebral edema (HACE). Hypoxia followed by reoxygenation (H/R) is also commonly used as a model to investigate pathology associated with ischemia/reperfusion as the latter condition is present in several disease states including stroke. In animal models, H/R has been shown to cause tight junction protein abnormalities, increased BBB paracellular permeability and edema. von Willebrand Factor (VWF) is a glycoprotein that is synthesized exclusively by endothelial cells and megakaryocytes. Endothelial cell-derived VWF is secreted constitutively and stored in Weibel-Palade bodies (WPB) from where it is released by regulated secretion into the plasma and subendothelium in response to endothelial activation. It has been demonstrated in vitro that exposure of cultured endothelial cells to hypoxia results in WPB exocytosis and VWF secretion. While it is known that VWF is expressed abundantly by cerebral endothelial cells, very little is known about the role of VWF in endothelial biology, particularly, in regulation of the BBB under stressful conditions. Several studies have shown that VWF protein is up regulated in plasma of patients with several neurological conditions involving BBB disruption such as stroke, severe head injury, cerebral malaria and cerebral venous sinus thrombosis. As it is known that C57BL/6 (wild-type) mice have increased BBB permeability induced by H/R, we investigated the status of BBB integrity in VWF-deficient mice (also on the C57BL/6 background). For these experiments, we used a mouse model of normobaric hypoxia (24 hours of 6% oxygen) followed by reoxygenation (1 hour ~21% oxygen). VWF antigen levels were measured by ELISA and BBB permeability was assessed by quantification of Evan's blue dye leakage into the brain. Our data indicate that plasma VWF levels in wild-type mice are significantly increased after hypoxia when compared to normoxic controls. Upon comparison with wild-type mice, we have determined that VWF-deficient mice have significantly less BBB permeability after H/R suggesting that VWF plays a role in BBB integrity under stressful conditions. We have previously reported that VWF-deficient mice have a defect in regulated P-selectin secretion (Denis et al., PNAS, 2001). To determine if the maintenance of BBB integrity found in VWF-deficient mice was due to lack of P-selectin we utilized an aptamer which inhibits P-selectin (Archemix). Inhibition of P-selectin in wild-type animals resulted in similar BBB permeability when compared to controls. Our findings suggest a critical role for VWF in BBB permeability after hypoxia/reoxygenation that is independent of P-selectin. Disclosures:No relevant conflicts of interest to declare.

PHYSIOLOGIC CHANGES IN COAGULATION AND FIBRINOLYSIS DURING NORMAL PREGNANCY

The human hemostatic system consists of multiple independent, yet integrally related, cellular and protein components that function to maintain blood fluidity under normal conditions and promote localized, temporary thrombus formation at sites of vascular injury . A normal hemostatic system is the human physiologic defense against exanguination. An abnormal hemostatic system can result in pathologic bleeding, vascular thrombosis, or both. The hemostatic system comprises of six major components: platelets, vascular endothelium , procoagulant plasma protein factors, natural anticoagulant proteins , fibrinolytic proteins, and antifibrinolytic proteins. Each of these six hemostatic components must be present in a fully functional form and in an adequate quantity to prevent excessive blood loss following vascular trauma and at the same time prevent pathologic thrombosis. The hemostatic system is highly regulated and maintains a delicate balance between a prohemorrhagic state and a prothrombotic state. Any significant acquired or congenital imbalance in the hemostatic scales can lead to a pathologic outcome. Alterations in the quantitative and qualatative status of any hemostatic cellular or protein element can have significant biologic effect. Platelet deficiency (thrombocytopenia), platelet adhesion defects, and platelet aggregation disorders are associated with an inability to form an adequate primary hemostatic platelet plug and can lead to significant mucocutaneous bleeding and post-traumatic, life-threatening hemorrhage. In contrast, a marked increase in platelet count (thrombocytosis) and accentuated platelet aggregation (sticky platelet syndrome) are associated with thromboembolic events. 32 Deficiency of a procoagulant factor integral to the intrinsic (factors XI, IX, and VIII), extrinsic (factor VII), or common (factors X, V, II and fibrinogen) pathway of coagulation is associated typically with a variable degree of bleeding tendency . Elevated levels of procoagulant factors, such as factor VIII, fibrinogen , and factor VII, however, are recognized risk factors for vascular disease. 28 , 47 Deficiency of natural anticoagulant proteins, such as protein C , protein S , antithrombin III (AT-III), and heparin cofactor II (HC-II), is associated with venous thromboembolic disease; a natural anticoagulant protein excess state associated with bleeding has not been described to date. 13 Deficiency of a fibrinolytic cascade component, such as tissue-type plasminogen activator (t-PA) or plasminogen; and excess plasma levels of the fibrinolytic inhibitor, plasminogen activator inhibitor-1 (PAI-1), have been linked to hypercoagulability and thrombosis. 24 , 29 , 53 Deficiency of fibrinolytic inhibitors, such as α-2 antiplasmin and PAI-1, may precipitate a hyperfibrinolytic bleeding state. 30 , 52 Deficiency of endothelial cell-derived von Willebrand factor is associated with altered primary and secondary hemostasis caused by deficient platelet anchoring at sites of vascular injury and shortened factor VIII survival characteristic of von Willebrand's disease . 20 Deficient endothelial cell production of thrombomodulin or release of t-PA may be associated with a thrombotic tendency. 19 The balance between these opposing groups of proteins and not the level of any individual factor seems most critical to hemostatic regulation. Pregnant women differ from nonpregnant adults in many physiologic and biochemical aspects. Normal pregnancy is associated with major changes in the coagulation and fibrinolytic systems . The many hemostatic derrangements detected during pregnancy must be considered to represent physiologic, adaptive, and preparatory mechanisms for the hemostatic challenges of delivery rather than pathologic processes. Most pregnant women do not manifest overt bleeding nor thrombotic problems. The hemostatic changes observed in each trimester of normal pregnancy and the puerperium reflect diverse changes in coagulation factor , fibrinolytic protein, regulatory protein , and cellular component activation, release, consumption, and synthesis. The fact that most hemostatic factors and proteins are liver derived, makes a review of coagulation alterations in pregnancy an appropriate component of an article on the liver and pregnancy. This article focuses on all hemostatic components involved in primary hemostasis (platelet plug formation) and secondary hemostasis (fibrin mesh formation) as well as the mechanisms responsible for thrombus localization (natural anticoagulants) and ultimate removal (fibrinolytic cascade proteins). The effects of each trimester of normal pregnancy and the puerperium on each of the major components of the hemostatic system are examined. Particular attention is paid to the balance between procoagulant and anticoagulant mechanisms and the net effect of the hemostatic changes at different periods during pregnancy.

Clearance of Normal and Type 2A von Willebrand Factor in the Rat

A model for the in vivo clearance of normal and mutant forms of human von Willebrand factor (vWF) has been established using catheterized rats. vWF clearance rates in rat plasma were determined by quantitation of reduced vWF subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and multimeric vWF was analyzed using nondenaturing SDS-agarose gels. Normal vWF derived from human umbilical vein endothelial cells displayed a biphasic pattern of clearance, with half times of 35 minutes (T 1/2 a; SD 15. min.) and 245 minutes (T 1/2 b; SD 76. min.); metabolic clearance rate = 0.65%/minute. High molecular weight multimers of vWF were cleared more rapidly than dimeric vWF. vWF containing the S1613P mutation found in some type 2A von Willebrand disease (vWD) patients was observed to undergo proteolysis in vivo resulting in a reduction of high molecular weight vWF and concomitant appearance of rapidly-migrating satellite species, although the overall clearance rate of vWF antigen was similar to wild type vWF. These results provide direct in vivo evidence that the S1613P mutation causes the characteristic type 2A vWD phenotype. Full-length recombinant vWF produced from transfected Chinese hamster ovary cells was cleared at a similar rate to endothelial cell-derived vWF, and recombinant vWF devoid of O-linked carbohydrates was cleared significantly faster. vWF devoid of sulfate was cleared at a similar rate as wild type vWF, indicating the sulfate moiety of vWF does not regulate in vivo clearance. This animal model should prove useful in subsequent in vivo analysis of additional forms of vWD and in the development of protease inhibitor therapy for 2A vWD.

Clearance of normal and type 2A von Willebrand factor in the rat

A model for the in vivo clearance of normal and mutant forms of human von Willebrand factor (vWF) has been established using catheterized rats. vWF clearance rates in rat plasma were determined by quantitation of reduced vWF subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and multimeric vWF was analyzed using nondenaturing SDS-agarose gels. Normal vWF derived from human umbilical vein endothelial cells displayed a biphasic pattern of clearance, with half times of 35 minutes (T 1/2 a; SD 15. min.) and 245 minutes (T 1/2 b; SD 76. min.); metabolic clearance rate = 0.65%/minute. High molecular weight multimers of vWF were cleared more rapidly than dimeric vWF. vWF containing the S1613P mutation found in some type 2A von Willebrand disease (vWD) patients was observed to undergo proteolysis in vivo resulting in a reduction of high molecular weight vWF and concomitant appearance of rapidly-migrating satellite species, although the overall clearance rate of vWF antigen was similar to wild type vWF. These results provide direct in vivo evidence that the S1613P mutation causes the characteristic type 2A vWD phenotype. Full-length recombinant vWF produced from transfected Chinese hamster ovary cells was cleared at a similar rate to endothelial cell-derived vWF, and recombinant vWF devoid of O-linked carbohydrates was cleared significantly faster. vWF devoid of sulfate was cleared at a similar rate as wild type vWF, indicating the sulfate moiety of vWF does not regulate in vivo clearance. This animal model should prove useful in subsequent in vivo analysis of additional forms of vWD and in the development of protease inhibitor therapy for 2A vWD.

Desmopressin Induces Adhesion of Normal Human Erythrocytes to the Endothelial Surface of a Perfused Microvascular Preparation

The interaction of red blood cells (RBCs) with vascular endothelium under flow conditions was investigated using the perfused rat mesocecum. Under videomicroscopy, normal human erythrocytes were found to adhere to the venular endothelium of desmopressin-treated microvasculature. Transmission electron microscopy showed that the erythrocytes were attached to the endothelial cells at discrete electron-dense sites. Compared with control preparations in which the microvasculature was perfused with Ringer's-albumin solution alone, more than a 10-fold increase in radioactivity was retained in the desmopressin-treated microvasculature when technetium (99mTc)-labeled erythrocytes were infused into the vasculature. This erythrocyte adherence was accompanied by a higher increment in vascular resistance during the passage of RBCs through the microcirculation, and by a delay in the recovery toward baseline. The erythrocyte adherence in desmopressin-treated microvasculature was completely abolished with antibodies to von Willebrand factor (vWF). Desmopressin infusion in rats resulted in elevated vWF antigen levels and the appearance of extra-large molecular weight forms of vWF in plasma. These findings suggest that normal erythrocytes adhere to desmopressin-conditioned microvascular endothelium and that endothelial cell-derived vWF is involved in the erythrocyte-endothelium interaction.

Desmopressin induces adhesion of normal human erythrocytes to the endothelial surface of a perfused microvascular preparation

The interaction of red blood cells (RBCs) with vascular endothelium under flow conditions was investigated using the perfused rat mesocecum. Under videomicroscopy, normal human erythrocytes were found to adhere to the venular endothelium of desmopressin-treated microvasculature. Transmission electron microscopy showed that the erythrocytes were attached to the endothelial cells at discrete electron-dense sites. Compared with control preparations in which the microvasculature was perfused with Ringer's-albumin solution alone, more than a 10-fold increase in radioactivity was retained in the desmopressin-treated microvasculature when technetium (99mTc)-labeled erythrocytes were infused into the vasculature. This erythrocyte adherence was accompanied by a higher increment in vascular resistance during the passage of RBCs through the microcirculation, and by a delay in the recovery toward baseline. The erythrocyte adherence in desmopressin-treated microvasculature was completely abolished with antibodies to von Willebrand factor (vWF). Desmopressin infusion in rats resulted in elevated vWF antigen levels and the appearance of extra-large molecular weight forms of vWF in plasma. These findings suggest that normal erythrocytes adhere to desmopressin-conditioned microvascular endothelium and that endothelial cell-derived vWF is involved in the erythrocyte-endothelium interaction.

Multimeric composition of endothelial cell-derived von Willebrand factor

The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC-conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.

Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases

We have previously found that the von Willebrand factor released by cultured human umbilical vein endothelial cells appeared as a single high molecular weight band in glyoxyl agarose electrophoresis. In the present studies we report that this high molecular weight endothelial cell-derived von Willebrand factor, when incubated with granulocyte lysates, was cleaved into a series of multimers indistinguishable from those seen in normal plasma (or type II von Willebrand disease). This von Willebrand factor-cleaving activity was released from granulocytes by calcium ionophore A23187 but was not detected in cytosolic fractions depleted of granular contents. It was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. This von Willebrand factor-cleaving activity thus provides a possible mechanism for the generation of plasma von Willebrand factor multimers from the high molecular weight form of von Willebrand factor secreted by endothelial cells.