[Severe bleeding in a patient with anti-c alloantibodies and a rare Rhesus phenotype treated with compatible erythrocyte concentrate from the blood bank of the Council of Europe]. / Ernstig bloedverlies bij een patient met anti-c-alloantistoffen en een bijzondere resusbloedgroep behandeld met compatibel erytrocytenconcentraat uit de bloedbank van de Raad van Europa.

An 84-year-old women had repeated gastrointestinal bleeding from a Dieulafoy lesion, i.e. a gastric or duodenal ulcer containing an aberrant artery. Her blood group was AB-D negative; her Rhesus phenotype was CCdee. In addition, antibody screening revealed anti-c alloantibodies as the result of a previous transfusion. Donors negative for D and c are very rare in Caucasian populations. Compatible red cell concentrates are available only from the European Bank of Frozen Blood of the Council of Europe, located at Sanquin in Amsterdam, Tthe Netherlands. The patient could be saved by requesting compatible erythrocyte concentrate from this blood bank. Severe blood loss poses a serious challenge in patients who are positive for alloantibodies against blood group antigens with a high frequency in the population, and in patients who are themselves negative for such antigens. The presence of alloantibodies is often the result of previous blood transfusions. In view of the large number of antigens on erythrocytes, one should therefore be conservative as to blood transfusion in order to prevent alloantibody formation.

Protein C inhibitor regulates the thrombin-thrombomodulin complex in the up- and down regulation of TAFI activation.

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a carboxy-peptidase B-like proenzyme that after activation by thrombin down regulates fibrinolysis. Thrombomodulin (TM) stimulates the activation of both TAFI and protein C whereas activated protein C (APC) inhibits the activation of TAFI by down regulating thrombin generation. Recently, protein C inhibitor (PCI) was identified as a potent inhibitor of thrombin bound to TM and it can thereby regulate the balance between TAFI activation, and inhibition of TAFI activation by APC. Both in a purified system and in plasma, activation of TAFI and protein C by [Ia-TM could be inhibited by PCI. Previously we found in plasma that at low concentrations (approximately 1 nM), TM predominantly stimulated the activation of TAFI whereas at higher concentrations of TM (approximately 10 nM) the activation of protein C resulted in inhibition of the activation of TAFI. In agreement with this. PCI inhibited the activation of TAFI at 1 nM TM whereas at 10 nM TM PCI inhibited the activation of protein C resulting in an increase in the activation of TAFI. This suggests that PCI can up regulate TAFI activation by inhibiting the protein C activation. PCI may therefore be an important regulator in the balance between coagulation and fibrinolysis by differentially inhibiting the activation of TAFI and of protein C. The local TM concentration plays an important role in the outcome of this process.

Characterization of transgenic mice that secrete functional human protein C inhibitor into the circulation.

Protein C inhibitor (PCI) is a heparin binding serine protease inhibitor in plasma, which exerts procoagulant activity by inhibiting thrombomodulin-bound thrombin or activated protein C (APC). Since the role of PCI in vivo is largely unknown we generated genetically modified mice with expression of human PCI mRNA in hepatocytes only. Three transgenic lines have been characterized. Transgenic mice did not show gross developmental abnormalities. Two lines showed a pericentral and one line showed a periportal expression pattern of human PCI mRNA in the liver. Genetically modified mice secreted a functional transgenic protein into the circulation (3-5 microg/ml plasma in heterozygous mice and 10 microg/ml in homozygous mice), which inhibited human APC activity in the presence of heparin. Interestingly, transgenic mice in which human PCI was expressed periportally in the liver had the highest specific activity. Endogenous mouse PCI mRNA could only be detected in the male and female reproductive system, but not in the liver, indicating that endogenous PCI levels in the circulation are low or even absent in mice. These results demonstrate that the human PCI transgenic mice are a suitable model for studying the in vivo role of PCI in blood coagulation.

Thrombomodulin activity of fat-derived microvascular endothelial cells seeded on expanded polytetrafluorethylene.

Lining the luminal surface of prosthetic vascular grafts with endothelial cells (cell seeding) will lower its thrombogenicity. Commonly used macrovascular human adult endothelial cells (HAEC) require in vitro cultivation before large enough numbers are obtained to cover grafts confluently. Fat-derived microvascular endothelial cells (MVEC) prove to be a good alternative as they can be harvested in much larger numbers while showing similar antithrombotic and fibrinolytic characteristics. An important anticoagulant function of macrovascular endothelial cells is due to the activity of thrombomodulin (TM) on their surface. In this study, the presence and functional activity of TM on fat-derived microvascular cells used in cell seeding was investigated. The expression and localization of TM on MVEC was studied using immunohistochemistry. Functional activity of TM on MVEC was measured by the generation of activated protein C (APC) and was compared to human umbilical vein endothelial cells (HUVEC). TM activity was studied in MVEC seeded on expanded polytetrafluorethylene (ePTFE) vascular prostheses and compared to blank prostheses. We found that TM was expressed on the surface of MVEC, both in vivo and vitro. TM-dependent generation of APC differed significantly between MVEC and HUVEC (3.98 +/- 1.2 vs. 3.0 +/- 0.7 nM, respectively). After seeding MVEC on vascular prostheses, TM activity did not change. APC generation was significantly higher on MVEC-seeded vascular grafts compared to blank grafts (4.0 +/- 0.7 vs. 1.7 +/- 0.5 nM, respectively). We conclude that TM is present and highly active on cultured MVEC. When seeded on ePTFE, MVEC retain the possibility to inhibit thrombin coagulant activity and to activate protein C. Therefore, since MVEC are readily available, the anticoagulant properties demonstrated here indicate that this cell type is suitable for cell seeding of vascular prostheses.

Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma.

Protein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited by PCI. The paradoxical inhibitory effect of PCI on both coagulant and anticoagulant proteases raised questions about the role of PCI in plasma. We studied the role of thrombomodulin (TM)-dependent inhibition of thrombin by PCI in a plasma system. Clotting was induced by addition of tissue factor to recalcified plasma in the absence or presence of TM, and clot formation was monitored using turbidimetry. In the absence of TM, PCI-deficient plasma showed a slightly shorter coagulation time compared with normal plasma. Reconstitution with a physiologic amount of PCI gave normal clotting times. Addition of PCI to normal plasma and protein C-deficient plasma resulted in a minor prolongation of the clotting time. This suggested that PCI can act as a weak coagulation inhibitor in the absence of TM. TM caused a strong anticoagulant effect in normal plasma due to thrombin scavenging and activation of the protein C anticoagulant pathway. This effect was less pronounced when protein C-deficient plasma was used, but could be restored by reconstitution with protein C. When PCI was added to protein C-deficient plasma in the presence of TM, a strong anticoagulant effect of PCI was observed. This anticoagulant effect was most likely caused by the TM-dependent thrombin inhibition by PCI. However, when PCI was added to normal plasma containing TM, a strong procoagulant effect of PCI was observed, due to the inhibition of protein C activation. PCI-deficient plasma was less coagulant in the presence of TM. A concentration-dependent increase in clotting time was observed when PCI-deficient plasma was reconstituted with PCI. The combination of these results suggest that the major function of PCI in plasma during coagulation is the inhibition of thrombin. A decreased generation of activated protein C is a procoagulant consequence of the TM-dependent thrombin inhibition by PCI. We conclude that TM alters PCI from an anticoagulant into a procoagulant during tissue factor-induced coagulation.

Protein C inhibitor may modulate human sperm-oocyte interactions.

Protein C inhibitor (PCI) is a heparin-binding plasma serine protease inhibitor that was originally identified as an inhibitor of activated protein C. PCI has a broad protease specificity, inhibiting several proteases in hemostasis and fibrinolysis by acting as a suicide substrate. Recently it has been reported that proteases of the reproductive system, such as acrosin, prostate-specific antigen, and tissue kallikrein, can also be effectively inhibited by PCI. However, a direct relation between PCI and physiological events during fertilization has not yet been established. An attempt was made to monitor and localize the inhibition of the sperm protease acrosin by PCI. Localization experiments for PCI on epididymal spermatozoa showed that PCI is present on the acrosomal cap of human spermatozoa, which demonstrates the early presence of PCI in the male reproductive tract. Induction of the acrosome reaction in ejaculated human spermatozoa resulted in the disappearance of PCI from the plasma membrane overlying the acrosomal head and the appearance of a strict distribution at the equatorial segment of human spermatozoa. The activity of acrosin in sperm extracts could be effectively inhibited by PCI. Zona-binding assays showed that active PCI is able to block sperm-egg binding in a concentration-dependent manner. The combination of the potent inhibition of acrosin and sperm-egg binding by PCI and the localization studies suggested that PCI may protect spermatozoa against premature acrosome reaction and degradation, thereby modulating the acrosin activity so that it can coincide with binding to the oocyte.

Role of the A+ helix in heparin binding to protein C inhibitor.

Interactions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A+ helix, is located at the N-terminus (residues 1-11), whereas the second region, the H helix, is positioned between residues 264 and 280 of PCI. Competition experiments with synthetic peptides based on the sequence of these regions demonstrated that the H helix has the highest affinity for heparin. In contrast to previous observations we found that the A+ helix peptide competed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide. Recombinant PCI was also used to investigate the role of the A+ helix in heparin binding. Full-length (wild-type) rPCI as well as an A+ helix deletion mutant of PCI (rPCI-delta 2-11) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A+ helix deletion mutant showed a decreased affinity for heparin in inhibition reactions with activated protein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A+ helix peptide competed with rPCI-delta w-11 for binding to heparin. This indicated that the interaction between PCI and heparin is fairly non-specific and that the interaction is primarily based on electrostatic interactions. In summary, our data suggest that the H helix of PCI is the main heparin binding region of PCI, but the A+ helix increases the overall affinity for the PCI-heparin interaction by contributing a second positively charged region to the surface of PCI.

Role of the H helix in heparin binding to protein C inhibitor.

Protein C inhibitor (PCI) is a plasma serine proteinase inhibitor (serpin) that is a major physiological regulator of activated protein C. Inhibition of its target proteinase is accelerated by heparin in a reaction that involves the binding of both inhibitor and proteinase to heparin to form a ternary complex. This study was undertaken to understand the role of the H helix region (residues 264-278) of PCI in heparin binding and used (i) a recombinant truncated PCI fusion protein of the first 294 residues, (ii) H helix synthetic peptides containing single Arg/Lys-->Glu substitutions, and (iii) site-directed Ala mutagenesis of 4 basic residues (Arg-269, Lys-270, Lys-276, and Lys-277) in the H helix region of full-length recombinant PCI (rPCI) expressed in Baculovirus. The PCI fusion protein interfered in heparin-accelerated PCI-proteinase inhibition reactions, and it bound to heparin-Sepharose. Compared to the wild-type PCI fusion protein, deletion of the H helix from the fusion protein resulted in a reduction of both heparin-Sepharose binding and the ability to compete for heparin during PCI-proteinase inhibition reactions. Competition assays with H helix synthetic peptides revealed that the R269E altered peptide was the least effective at blocking heparin-catalyzed PCI-proteinase inhibition reactions. Compared with full-length active wild-type rPCI, R269A: K270A and K276A:K277A rPCI both had reduced heparin-Sepharose binding, but only R269A:K270A rPCI showed a loss of heparin-accelerated proteinase inhibition for both activated protein C and thrombin. We conclude that a major heparin-binding site of PCI is the H helix, unlike its heparin-binding serpin homologues antithrombin and heparin cofactor II, which bind heparin primarily through the D helix.