Scott T. Cooper

Protein C Inhibitor Is a Potent Inhibitor of the Thrombin-Thrombomodulin Complex

Protein C inhibitor (PCI), a plasma serine protease inhibitor, inhibits several proteases including the anticoagulant enzyme, activated protein C (APC), and the coagulation enzymes, thrombin and factor Xa. Previous studies have shown that thrombin and APC are inhibited at similar rates by PCI and that heparin accelerates PCI inhibition of both enzymes more than 20-fold. We now demonstrate that the thrombin-binding proteoglycan, rabbit thrombomodulin, accelerates inhibition of thrombin by PCI ≈140-fold (k = 2.4 × 10 in the presence of TM compared to 1.7 × 10M s in the absence of TM). Most of this effect is mediated by protein-protein interactions since the active fragment of TM composed of epidermal growth factor-like domains 4-6 (TM 4-6) accelerates inhibition by PCI ≈59-fold (k = 1.0 × 10M s). The mechanism by which TM alters reactivity with PCI appears to reside in part in an alteration of the S2 specificity pocket. Replacing Phe with Pro at the P2 position in the reactive loop of PCI yields a mutant that inhibits thrombin better in the absence of TM (k = 6.3 × 10M s), but TM 4-6 enhances inhibition by this mutant ≈9-fold (k = 5.8 × 10M s) indicating that TM alleviates the inhibitory effect of the less favored Phe residue. These results indicate that PCI is a potent inhibitor of the protein C anticoagulant pathway at the levels of both zymogen activation and enzyme inhibition.

Vascular Localization of the Heparin-binding Serpins Antithrombin, Heparin Cofactor II, and Protein C Inhibitor

Heparin is one of the most widely used drugs in the world, acting as an anticoagulant by stimulating the reaction between heparin-binding serpins and the serine proteases of the coagulation cascade. To determine whether the heparin-binding serpins antithrombin (AT), heparin cofactor II (HCII), and protein C inhibitor (PCI) were bound to glycosaminoglycans on the endothelial wall, a bolus of heparin (100 U/kg body weight) was in jected into human volunteers, and serpin concentrations and activities were measured in both pre- and postheparin plasma. No increase in circulating concentrations of AT, HCII, or PCI were observed in postheparin plasma. Sim ilarly, AT and HCII activities did not increase in posthe parin plasma. In contrast, the concentration of another heparin-binding protein, lactoferrin (LF), increased six- fold after heparin injection. Immunohistochemistry of hu man artery was performed using polyclonal antisera to AT, HCII, PCI, LF, and tissue factor pathway inhibitor (TFPI), another heparin-binding protein released by hep arin injection. AT, HCII, and PCI were present in the intima, whereas LF, TFPI, and traces of AT were found on the surface of the vessel wall. The distribution of the proteins in the vessel wall supports the results of the hep arin-injection studies and may give valuable clues to the role of each protein in vascular homeostasis.

Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin.

Thrombomodulin (TM), or its epidermal growth factor-like domains 456 (TM456), enhances the catalytic efficiency of thrombin toward both protein C and protein C inhibitor (PCI) by 2–3 orders of magnitude. Structural and mutagenesis data have indicated that the interaction of basic residues of the heparin-binding exosite of protein C with the acidic residues of TM4 is partially responsible for the efficient activation of the substrate by the thrombin-TM456 complex. Similar to protein C, PCI has a basic exosite (H-helix) that constitutes the heparin-binding site of the serpin. To determine whether TM accelerates the reactivity of thrombin with PCI by providing a binding site for the H-helix of the serpin, an antithrombin (AT) mutant was constructed in which the H-helix of the serpin was replaced with the same region of PCI (AT-PCIH-helix). Unlike PCI, the H-helix of AT is negatively charged. It was discovered that TM456 slightly (<2-fold) impaired the reactivity of AT with thrombin; however, it enhanced the reactivity of AT-PCIH-helix with the protease by an order of magnitude. Further studies revealed that the substitution of Arg35 of thrombin with an Ala also resulted in an order of magnitude enhancement in reactivity of the protease with both PCI and AT-PCIH-helix independent of TM. We conclude that TM enhances the reactivity of PCI with thrombin by providing both a binding site for the serpin and a conformational modulation of the extended binding pocket of thrombin.

Basic residues in the 37-loop of activated protein C modulate inhibition by protein C inhibitor but not by α1-antitrypsin

The role of lysines 37-39 (chymotrypsin numbering) in the 37-loop of the serine protease activated protein C (APC) was studied by expressing acidic and neutral recombinant APC (rAPC) mutants. Activity of the APC mutants was assessed using human plasma and plasma-purified and recombinant derivatives of protein C inhibitor (PCI; also known as plasminogen activator inhibitor-3) and alpha(1)-antitrypsin, with and without heparin. The catalytic properties of the mutants to small peptidyl substrates were essentially the same as wild-type rAPC (wt-rAPC), yet their plasma anticoagulant activities were diminished. Analysis of the rAPC-protease inhibitor complexes formed after addition of wt-rAPC and mutants to plasma revealed no change in the inhibition pattern by alpha(1)-antitrypsin but a reduction in mutant complex formation by PCI in the presence of heparin. Using purified serpins, we found that inhibition rates of the mutants were the same as wt-rAPC with alpha(1)-antitrypsin; however, PCI (plasma-derived and recombinant forms) inhibition rates of the acidic mutants were slightly faster than that of wt-rAPC without heparin. By contrast, PCI-heparin inhibition rates of the mutants were not substantially accelerated compared to wt-rAPC. The mutants had reduced heparin-binding properties compared to wt-rAPC. Molecular modeling of the PCI-APC complex with heparin suggests that heparin may function not only to bridge PCI to APC, but also to alleviate putative non-optimal intermolecular interactions. Our results suggest that the basic residues of the 37-loop of APC are involved in macromolecular substrate interactions and in heparin binding, and they influence inhibition by PCI (with or without heparin) but not by alpha(1)-antitrypsin, two important blood plasma serpins.

Assessment of the Interaction Between Urokinase and Reactive Site Mutants of Protein C Inhibitor

Urokinase-type plasminogen activator (uPA) is a serine protease involved in pericellular proteolysis and tumor cell metastasis via plasmin-mediated degradation of extracellular matrix proteins. Plasma uPA is inhibited by the serine protease inhibitor protein C inhibitor (PCI) by the insertion of PCIs reactive site loop into the active site of the protease. To better understand the structural aspects of this inhibition, 15 reactive-site mutants of recombinant PCI (rPCI) were assayed for differences in uPA inhibition. These assays revealed that substitutions at the P1 Arg354 and P3 Thr352 sites of rPCI were detrimental to inhibitory activity, while P3′ Arg357 mutations had little effect upon the inhibition rate. However, replacement of the P2 Phe353 with small residues like Ala and Gly increased the effectiveness of rPCI three- to four fold. To explain these altered rates of inhibition, a computer-derived molecular model of uPA was generated and docked to a model of PCI to simulate complex formation. The changes made by mutagenesis were then recreated in the model of uPA–PCI. In accordance with the kinetic data, the poor performance of P3 variants is primarily attributable to charge repulsion, while alleviation of steric hindrance at P2 produces the observed increase in uPA inhibition. In the model, residues at P3′ interact with PCI rather than uPA, consistent with P3′ variants demonstrating that little variation from wild-type activity. Ultimately, this combination of mutagenesis and molecular modeling will further refine our understanding of the interaction between PCI and uPA.

The hibernating 13-lined ground squirrel as a model organism for potential cold storage of platelets

Hibernating mammals have developed many physiological adaptations to extreme environments. During hibernation, 13-lined ground squirrels (Ictidomys tridecemlineatus) must suppress hemostasis to survive prolonged body temperatures of 4-8°C and 3-5 heartbeats per minute without forming lethal clots. Upon arousal in the spring, these ground squirrels must be able to quickly restore normal clotting activity to avoid bleeding. Here we show that ground squirrel platelets stored in vivo at 4-8°C were released back into the blood within 2 h of arousal in the spring with a body temperature of 37°C but were not rapidly cleared from circulation. These released platelets were capable of forming stable clots and remained in circulation for at least 2 days before newly synthesized platelets were detected. Transfusion of autologous platelets stored at 4°C or 37°C showed the same clearance rates in ground squirrels, whereas rat platelets stored in the cold had a 140-fold increase in clearance rate. Our results demonstrate that ground squirrel platelets appear to be resistant to the platelet cold storage lesions observed in other mammals, allowing prolonged storage in cold stasis and preventing rapid clearance upon spring arousal. Elucidating these adaptations could lead to the development of methods to store human platelets in the cold, extending their shelf life.

Integrating bioinformatics into undergraduate courses

Bioinformatics has become an essential tool in many aspects of biochemistry and molecular biology research. Unfortunately, it has lagged behind in biochemistry and molecular biology education, often because of limitations i n available computers, software, and training of educators. Due to these limitations, bioinformatics courses have often been taught at the graduate level, with a n occasional undergraduate lecture or lab incorporating a bioinformatics unit. With the current explosion in genomic and proteomic data it has become as important to train undergraduates in bioinformatics as it was to train them in column chromatography or electrophoresis in the recent past. In the past few years, easy to use, web-based programs such as Biology Workbench [I] and Protein Explorer [2] have lowered the hurdles that often kept bioinformatics out of undergraduate courses. Both of the programs are free, and students can access them from a n y computer with internet access. This enables teachers to assign homework and gives students access to their data while writing lab reports. Biology Workbench is also novel in that students are allowed to store their sequence data on a server. This has been especially useful i n that students do not have to carry their data with them on disks which can be forgotten or lost. Protein Explorer runs over the Netscape plug-in Chime and is similar to the popular program RasMol. However, instead of entering commands on a command line as in RasMol, Protein Explorer uses a menu that students can click, letting them focus on the protein structure and not on memorizing commands. Finally, Protein Explorer allows students to compare multiple amino acid sequence alignments in three dimensions by superimposing the alignment on a known crystal structure of one of the proteins in the alignment. We have used two approaches to train undergraduates in the basic concepts of bioinformatics at the University of Wisconsin-La Crosse (UWL). The first approach is to integrate bioinformatics into existing courses including Biochemistry, Genetics, Microbial

Effects of hibernation on bone marrow transcriptome in thirteen-lined ground squirrels.

Mammalian hibernators adapt to prolonged periods of immobility, hypometabolism, hypothermia, and oxidative stress, each capable of reducing bone marrow activity. In this study bone marrow transcriptomes were compared among thirteen-lined ground squirrels collected in July, winter torpor, and winter interbout arousal (IBA). The results were consistent with a suppression of acquired immune responses, and a shift to innate immune responses during hibernation through higher complement expression. Consistent with the increase in adipocytes found in bone marrow of hibernators, expression of genes associated with white adipose tissue are higher during hibernation. Genes that should strengthen the bone by increasing extracellular matrix were higher during hibernation, especially the collagen genes. Finally, expression of heat shock proteins were lower, and cold-response genes were higher, during hibernation. No differential expression of hematopoietic genes involved in erythrocyte or megakaryocyte production was observed. This global view of the changes in the bone marrow transcriptome over both short term (torpor vs. IBA) and long term (torpor vs. July) hypothermia can explain several observations made about circulating blood cells and the structure and strength of the bone during hibernation.

Essential thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin

Summary.  Background: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin. Objective: Fifty‐five Ala‐scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. Results: Residues around the active site (Tyr50 and Glu202) and the sodium‐binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite‐2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin‐accelerated inhibition of thrombin by PCI. Exosite‐1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin–TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the ∼150‐fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite‐2 mutants that were essential for heparin‐catalyzed PCI–thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin–TM. Conclusions: Collectively, these results show that (i) similar thrombin exosite‐2 residues are critical for the heparin‐catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin–TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin–TM.

Inhibition of a thrombin anion-binding exosite-2 mutant by the glycosaminoglycan-dependent serpins protein C inhibitor and heparin cofactor II

Antithrombin (ATIII), heparin cofactor II (HCII) and protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) are serine protease inhibitors (serpins) whose thrombin inhibition activity is accelerated in the presence of glycosaminoglycans. We compared the inhibition properties of PCI and HCII to ATIII using R93A/R97A/R101A thrombin, an anion-binding exosite-2 (exosite-2) mutant that has greatly reduced heparin-binding properties. Heparin-enhanced PCI inhibition of R93A/R97A/R101A thrombin was only approximately 2-fold compared to 40-fold enhancement with wild-type recombinant thrombin. Thrombomodulin (TM) (with or without the chondroitin sulfate moiety) accelerated PCI inhibition of both wild-type and R93A/R97A/R101A thrombins. HCII achieved the same maximum activity in the presence of heparin with both wild-type and R93A/R97A/R101A thrombins; however, the optimum heparin concentration was 20 times greater than the reaction with wild-type thrombin, indicative of a decrease in heparin affinity. Dermatan sulfate (DSO4)-catalyzed HCII thrombin inhibition was unchanged in R93A/R97A/R101A thrombin compared to wild-type recombinant thrombin. These results suggest that PCI is similar to ATIII and depends upon ternary complex formation with heparin and these specific thrombin exosite-2 residues to accelerate thrombin inhibition. In contrast, HCII does not require Arg(93), Arg(97) and Arg(101) of thrombin exosite-2 and further supports the hypothesis that HCII uses an allosteric process following glycosaminoglycan binding to inhibit thrombin.