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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) approximately 60 nM). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC(50) approximately 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 of (125)I-labeled FVIII by LRP-expressing cells ( approximately 8 fmol/10(5) 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) approximately 5 x 10(4) M(-1) s(-1); k(off) approximately 2.5 x 10(-3) s(-1); K(d) approximately 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 the in vivo survival of FVIII.

Cultured bovine aortic endothelial cells are associated with an unusually stable fibrinolytic inhibitor (Loskutoff, D.J., van Mourik, J.A., Erickson, L.A., and Lawrence, D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2956-2960). This inhibitor was purified to apparent homogeneity from medium conditioned by these cells by a combination of concanavalin A affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a single-chain glycoprotein of apparent Mr 50,000 +/- 2,500 and isoelectric point of 4.5-5.0, and inhibits the ability of both urokinase and tissue-type plasminogen activator to cleave and active plasminogen. This inhibition of plasminogen activator activity is associated with the formation of an enzyme-inhibitor complex which can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified inhibitor retains full activity after incubation in the presence of 0.1% sodium dodecyl sulfate, or at pH 2.7, two treatments which rapidly destroy the activity of protease nexin, another cellular inhibitor of fibrinolysis. The inhibitor purified from cloned endothelial cells cultured in the presence of L-[3,4,5-3H]leucine represented 2.5-12% of the total radiolabeled protein released by the cells in a 24-h period. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a protein which inhibits plasminogen activators and is distinct from protease nexin. It is a major endothelial cell product, and, as such, probably plays an important role in regulating the fibrinolytic system of these cells.

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.

To define the role of membrane components that function in endothelial cell physiology and to characterize them biochemically, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained producing antibodies which bound to endothelial cells and also to platelets. The antibody of one of these clones, CLB-HEC 75, was studied in more detail. This antibody is directed against a single protein which is synthesized constitutively by endothelial cells and is expressed on the surface of both endothelial cells and platelets. The CLB-HEC 75 antigen was isolated from Nonidet P-40-solubilized endothelial cells and platelets by immunoprecipitation and exhibited an apparent molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 145,000 in the presence of 2-mercaptoethanol. Two-dimensional polyacrylamide gel electrophoresis and crossed immunoelectrophoresis revealed that the mobility of the CLB-HEC 75 antigen relative to platelet glycoproteins Ib, IIa, IIb, and IIIa fits previously defined criteria for platelet membrane glycoprotein IIa. The CLB-HEC 75 antigen isolated from endothelial cells co-migrated under all conditions tested with the antigen from platelets. These results indicate that endothelial cells share a plasma membrane protein indistinguishable from platelet membrane glycoprotein IIa. This protein may be a component involved in the interaction of endothelial cells with their environment including coagulation factors, platelets, and the subendothelial matrix. CLB-HEC 75 may serve as a useful tool for studying these processes.

In previous studies have shown that the interaction between factor IXa and VIII involves the light chain of factor VIII and that this interaction inhibited by the monoclonal antibody CLB-CAg A against the factor VIII region Gln1778-Asp1840 (Lenting, P.J., Donath, M.J.S.H., van Mourik, J.A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu1801 and Met1823. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the factor VII region Tyr1786-Ala1834. Factor IXa binding was found to be particularly efficient to peptide corresponding to the factor VIII sequences Lys1804-Lys1818 and Glu1811-Gln1820. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys1804-Lys1818 and Glu1811-Gln1820 interfere with binding of factor IXa to immobilized factor VIII light chain (Ki approximately 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit factor X activation by factor IXa in the presence of factor VIIIa (Ki approximately 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the factor IX zymogen and its activated form, factor IXa, with the same affinity (apparent Kd approximately 0.2 mM), whereas the complete factor VIII light chain displays preferential binding to factor IXa. In conclusion, our results demonstrate that peptides consisting of the factor VIII light chain residues Lys1804-Lys1818 and Glu1811-Gln1820 share a factor IXa binding site that is essential for the assembly of the factor X-activating factor IXa-factor VIIIa complex. We propose that the overlapping sequence Glu1811-Lys1818 comprises the minimal requirements for binding to activated factor IX.

The endothelial cell-type plasminogen activator inhibitor (PAI-1) may exist in an inactive, latent form that can be converted into an active form upon treatment of the protein with denaturants, such as sodium dodecyl sulfate, guanidine HCl, or urea. The present paper demonstrates that latent PAI-1 can be activated by lipid vesicles containing the negatively charged phospholipids phosphatidylserine (PS) or phosphatidylinositol. The presence of a net negative charge on the phospholipid headgroup is essential for activation, since lipid vesicles consisting exclusively of zwitterionic phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, do not activate PAI-1. In the presence of PS vesicles, PAI-1 inhibited tissue-type plasminogen activator 50-fold more effectively than in the absence of phospholipids, whereas sodium dodecyl sulfate enhanced PAI-1 activity by 25-fold. In mixed phospholipid vesicles containing PS and phosphatidylcholine in various molar ratios, the extent of PAI-1 activation was directly related to the PS content of the phospholipid membrane. Ca2+ ions interfered with the inhibitory activity of PS-activated PAI-1, suggesting that Ca2+ ions may regulate PAI-1 activity in the presence of negatively charged phospholipids. An important consequence of these findings is that, as in blood coagulation, negatively charged phospholipids may play an important regulatory role in controlling the fibrinolytic system by activating an inhibitor of tissue-type plasminogen activator.

The role of Factor VIII light chain cleavage in Factor VIII activation and subunit interaction was investigated. Purified Factor VIII was dissociated into its separate subunits, and the isolated light chain was cleaved by thrombin at position Arg1689 or by Factor Xa at position Arg1721. These Factor VIII light chain derivatives then were used for reconstitution with purified Factor VIII heavy chain to obtain heterodimers that were exclusively cleaved within the light chain. Intact and cleaved light chain could effectively be reassociated with heavy chain, with concomitant regain of Factor VIII cofactor function. The association rate constant of Factor Xa-cleaved light chain was found to be 3-fold lower than that of thrombin-cleaved or intact light chain, suggesting a role of the region Ser1690-Arg1721 in subunit assembly. Dissociation rate constants, however, were independent of Factor VIII light chain cleavage. Low ionic strength was observed to promote association but to destabilize the Factor VIII heterodimer. At high ionic strength, Factor VIII dissociation was extremely slow (kappa off approximately 10(-5) s-1) for all Factor VIII light chain derivatives, indicating that Factor VIII light chain cleavage is not related to Factor VIII dissociation. Furthermore, Factor VIII light chain cleavage does not affect enzyme-cofactor assembly, since the various light chain derivatives proved equally efficient in binding to Factor IXa (Kd approximately 15 nM). Studies in a purified Factor X-activating system demonstrated that thrombin and Factor Xa activate Factor VIII to the same extent. However, Factor Xa differed from thrombin in that it cleaved at Arg1721 rather than at Arg1689. Reassociated heterodimers of Factor VIII heavy chain and intact light chain did not promote Factor X activation. In contrast, heterodimers that contained cleaved light chain exhibited substantial Factor VIIIa activity. These data demonstrate that a single cleavage at either Arg1689 or Arg1721 converts the inactive Factor VIII heterodimer into an active cofactor of Factor IXa.

The transition of the factor IX zymogen into the enzyme factor IXa beta was investigated. For this purpose, the activation intermediate factors IX alpha and IXa alpha were purified after cleavage of the Arg145-Ala146 and Arg180-Val181 bonds, respectively. These intermediates were compared for a number of functional properties with factor IXa beta, which is cleaved at both positions. Factor IXa alpha was equal to factor IXa beta in hydrolyzing the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide (kcat/Km approximately 120 s-1 M-1) but was less efficient in factor X activation. Factor IX alpha was incapable of generating factor Xa but displayed reactivity toward p-nitrophenol p-guanidinobenzoate and the peptide substrate. The catalytic efficiency, however, was 4-fold lower compared with factor IXa alpha and factor IXa beta. Factor IX alpha and factor IXa beta had similar affinity for the inhibitor benzamidine (Ki approximately 2.5 mM), and amidolytic activity of both species was inhibited by Glu-Gly-Arg-chloromethyl ketone and antithrombin III. Unlike factor IXa beta, factor IX alpha was unable to form SDS stable complexes with antithrombin III. Moreover, inhibition of factor IXa beta and factor IX alpha by Glu-Gly-Arg-chloromethyl ketone followed distinct pathways, because factor IX alpha was inhibited in a nonirreversible manner and displayed only minor incorporation of the dansylated inhibitor into its catalytic site. These data demonstrate that the catalytic site of factor IX alpha differs from that of the fully activated factor IXa beta. Factor IX and its derivatives were also compared with regard to complex assembly with factor VIII in direct binding studies employing the immobilized factor VIII light chain. Factor IX alpha and factor IXa beta displayed a 30-fold higher affinity for the factor VIII light chain (Kd approximately 12 nM) than the factor IX zymogen. Factor IXa alpha showed lower affinity (Kd approximately 50 nM) than factor IX alpha and factor IXa beta, which may explain the lower efficiency of factor X activation by factor IXa alpha. Collectively, our data indicate that cleavage of the Arg180-Val181 bond develops full amidolytic activity but results in suboptimal binding to the factor VIII light chain. With regard to cleavage of the Arg145-Ala146 bond, we have demonstrated that this results in the transition of the factor IX zymogen into an enzyme that lacks proteolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)