Human semen contains very potent blood clotting activity; for example, seminal serum diluted up to 10,000-fold significantly decreased the recalcification clotting time of blood plasma. This seminal coagulant activity was dependent on factor X and calcium ions, suggesting the presence of a facto X activator. Immunoblotting analysis and immunoadsorption studies confirmed the presence of tissue factor antigen (45 kDa) in semen. Centrifugation studies suggested that tissue factor was membrane associated, and fractionation of seminal serum by gel filtration followed by immunoelectron microscopy revealed that tissue factor antigen was on the prostasome vesicle surface. Tissue factor originated from prostatic fluid and not from seminal vesicle secretions. Tissue factor antigen averaged 21 ng/ml in seminal serum. Hypothetical roles for very high levels of tissue factor in semen include several possibilities. In the event of abrasion and bleeding during intercourse, rapid blood clotting at lesion sites would prevent sperm and seminal components, including infectious agents such as human immunodeficiency virus, from entering the blood stream, generating antibodies, or promoting infectious disease. This could imply that development of infection from semen-borne agents or development of antisperm antibodies in some patients could result from impairment or absence of seminal tissue factor.

Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2-4-fold. This correlated with 1.3-1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7-13-fold decrease in affinity for LRP and 1.6-2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an approximately 4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by approximately 1.9 and approximately 3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.

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Summary. Background: The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664-C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. Aims: Here, we characterize the functional parameters of C664-C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662-C1828 FVIII). Methods: In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. Results: Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662-C1828 FVIIIa had normal activity while C664-C1826 FVIIIa had reduced activity. Both C664-C1826 FVIIIa and C662-C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. Conclusion: Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.

Human protein C-inhibitor (PCI) was isolated from human citrated plasma by combining rivanol precipitation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on dextran sulfate Sepharose. The purified PCI migrated with the ß-globulins and was free from protein contaminations as judged by immunoelectrophoresis. In SDS-PAGE under reducing and unreducing conditions PCI showed a single band at M t = 57 000. The specific activity of the inhibitor was 226 units/mg. Surprisingly, the isolated PCI inhibited the amidolytic activity of urokinase (u-PA) on '-olu-Gly--Arg-pNA (S-2444) in a time-dependent manner. Heparin, dextran sulfate and pentosanpolysulfate accelerated the reaction catalytically. PCI revealed itself as a non-competitive inhibitor of u-PA. The Rvalue was determined to be 7.9 10~8M. Inhibition of amidolytic activity was found to be associated with the formation of an 1:1 equimolar complex with a M r of 110 000 as demonstrated by means of polyacrylamide gel electrophoresis and following Western blotting technique using polyclonal antibodies against u-PA and PCI. The specific activity of the isolated PCI of 226 units/mg, which approximates the theoretical value of pure PCI, indicates a highly purified preparation of PCI. The heparin-dependent inhibition of urokinase by this highly purified protein as well as comparison of the kinetic data and amino-acid composition of both PCI and the recently described plasminogen activator inhibitor (PAI) 3 give high evidence of identity of PCI and PAI-3.

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6. Dahlbiick H, Carlsson M, Svcnsson I'J. Familial thrombophilia due to a pre viously unrecognized mechanism characterized by poor anticoagulant res ponse to activated protein (': Prediction of a cofactor to activated protein ('.