Type 2 Diabetes Coagulopathy Proteins May Conflict With Biomarkers Reflective of COVID-19 Severity.

Objective: Detailed proteomic analysis in a cohort of patients with differing severity of COVID-19 disease identified biomarkers within the complement and coagulation cascades as biomarkers for disease severity has been reported; however, it is unclear if these proteins differ sufficiently from other conditions to be considered as biomarkers. Methods: A prospective, parallel study in T2D (n = 23) and controls (n = 23). A hyperinsulinemic clamp was performed and normoglycemia induced in T2D [4.5 ± 0.07 mmol/L (81 ± 1.2 mg/dl)] for 1-h, following which blood glucose was decreased to ≤2.0 mmol/L (36 mg/dl). Proteomic analysis for the complement and coagulation cascades were measured using Slow Off-rate Modified Aptamer (SOMA)-scan. Results: Thirty-four proteins were measured. At baseline, 4 of 18 were found to differ in T2D versus controls for platelet degranulation [Neutrophil-activating peptide-2 (p = 0.014), Thrombospondin-1 (p = 0.012), Platelet factor-4 (p = 0.007), and Kininogen-1 (p = 0.05)], whilst 3 of 16 proteins differed for complement and coagulation cascades [Coagulation factor IX (p < 0.05), Kininogen-1 (p = 0.05), and Heparin cofactor-2 (p = 0.007)]; STRING analysis demonstrated the close relationship of these proteins to one another. Induced euglycemia in T2D showed no protein changes versus baseline. At hypoglycemia, however, four proteins changed in controls from baseline [Thrombospondin-1 (p < 0.014), platelet factor-4 (p < 0.01), Platelet basic protein (p < 0.008), and Vitamin K-dependent protein-C (p < 0.00003)], and one protein changed in T2D [Vitamin K-dependent protein-C, (p < 0.0002)]. Conclusion: Seven of 34 proteins suggested to be biomarkers of COVID-19 severity within the platelet degranulation and complement and coagulation cascades differed in T2D versus controls, with further changes occurring at hypoglycemia, suggesting that validation of these biomarkers is critical. It is unclear if these protein changes in T2D may predict worse COVID-19 disease for these patients. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT03102801.

Serum biomarker analysis in patients with premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a primary ovarian defect characterized by premature depletion of ovarian follicles before 40 years of age. The disorder has been attributed to various causes, but the study of altered proteins in serum levels as the cause is rare. Additionally, identifying novel biomarkers can contribute to more accurate diagnosis or prognosis of POI. In the present study, a solid-phase antibody array simultaneously detecting multiple proteins was used to analyze POI serum with menopausal and healthy fertile subjects as control groups. As a result, compared to the menopause and healthy fertile groups, eleven proteins, including Neurturin, Frizzled-5, Serpin D1, MMP-7, ICAM-3, IL-17F, IFN-gamma R1, IL-29, IL-17R, IL-17C and Soggy-1, were uniquely down-regulated, and Afamin was particularly up-regulated in POI serum. More importantly, all of these factors were firstly found to be associated with POI in this study, suggesting that these proteins may participate in the pathogenesis of POI and may be novel serum biomarkers for POI.

Recombinant dermatan sulfate is a potent activator of heparin cofactor II-dependent inhibition of thrombin.

The glycosaminoglycan dermatan sulfate (DS) is a well-known activator of heparin cofactor II-dependent inactivation of thrombin. In contrast to heparin, dermatan sulfate has never been prepared recombinantly from material of non-animal origin. Here we report on the enzymatic synthesis of structurally well-defined DS with high anticoagulant activity. Using a microbial K4 polysaccharide and the recombinant enzymes DS-epimerase 1, dermatan 4-O-sulfotransferase 1, uronyl 2-O-sulfotransferase and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, several new glycostructures have been prepared, such as a homogenously sulfated IdoA-GalNAc-4S polymer and its 2-O-, 6-O- and 2,6-O-sulfated derivatives. Importantly, the recombinant highly 2,4-O-sulfated DS inhibits thrombin via heparin cofactor II, approximately 20 times better than heparin, enabling manipulation of vascular and extravascular coagulation. The potential of this method can be extended to preparation of specific structures that are of importance for binding and activation of cytokines, and control of inflammation and metastasis, involving extravasation and migration.

Analysis of the gene expression profile in response to human epididymis protein 4 in epithelial ovarian cancer cells.

Currently, there are emerging multiple studies on human epididymis protein 4 (HE4) in ovarian cancer. HE4 possesses higher sensitivity and specificity than CA125 in the confirmative early diagnosis for ovarian cancer. Although much attention has been given to explore its clinical application, research of the basic mechanisms of HE4 in ovarian cancer are still unclear. In the present study, we provide fundamental data to identify full-scale differentially expressed genes (DEGs) in response to HE4 by use of human whole-genome microarrays in human epithelial ovarian cancer cell line ES-2 following overexpression and silencing of HE4. We found that a total of 717 genes were upregulated and 898 genes were downregulated in the HE4-overexpressing cells vs. the HE4-Mock cells, and 166 genes were upregulated and 285 were downregulated in the HE4-silenced cells vs. the HE4-Mock cells. An overlap of 16 genes consistently upregulated and 8 genes downregulated in response to HE4 were noted. These DEGs were involved in MAPK, steroid biosynthesis, cell cycle, the p53 hypoxia pathway, and focal adhesion pathways. Interaction network analysis predicted that the genes participated in the regulatory connection. Highly differential expression of the FOXA2, SERPIND1, BDKRD1 and IL1A genes was verified by quantitative real-time PCR in 4 cell line samples. Finally, SERPIND1 (HCII) was validated at the protein level by immunohistochemistry in 107 paraffin-embedded ovarian tissues. We found that SERPIND1 may act as a potential oncogene in the development of ovarian cancer. The present study displayed the most fundamental and full-scale data to show DEGs in response to HE4. These identified genes may provide a theoretical basis for investigations of the underlying molecular mechanism of HE4 in ovarian cancer.

Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development.

Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

Loss of Biglycan Enhances Thrombin Generation in Apolipoprotein E-Deficient Mice: Implications for Inflammation and Atherosclerosis.

OBJECTIVE: Thrombin signaling promotes atherosclerosis by initiating inflammatory events indirectly through platelet activation and directly via protease-activated receptors. Therefore, endogenous thrombin inhibitors may be relevant modulators of atheroprogression and cardiovascular risk. In addition, endogenous thrombin inhibitors may affect the response to non-vitamin K-dependent oral anticoagulants. Here, the question was addressed whether the small leucine-rich proteoglycan biglycan acts as an endogenous thrombin inhibitor in atherosclerosis through activation of heparin cofactor II. APPROACH AND RESULTS: Biglycan concentrations were elevated in the plasma of patients with acute coronary syndrome and in male Apolipoprotein E-deficient (ApoE(-/-)) mice. Biglycan was detected in the glycocalyx of capillaries and the subendothelial matrix of arterioles of ApoE(-/-) mice and in atherosclerotic plaques. Thereby a vascular compartment is provided that may mediate the endothelial and subendothelial activation of heparin cofactor II through biglycan. ApoE and Bgn double-deficient (ApoE(-/-)/Bgn(-/0)) mice showed higher activity of circulating thrombin, increased platelet activation and platelet adhesion in vivo, supporting a role of biglycan in balancing thrombin activity. Furthermore, concentrations of circulating cytokines and aortic macrophage content were elevated in ApoE(-/-)/Bgn(-/0) mice, suggesting a proinflammatory phenotype. Elevated platelet activation and macrophage accumulation were reversed by treating ApoE(-/-)/Bgn(-/0) mice with the thrombin inhibitor argatroban. Ultimately, ApoE(-/-)/Bgn(-/0) mice developed aggravated atherosclerosis. CONCLUSIONS: The present results indicate that biglycan plays a previously unappreciated protective role during the progression of atherosclerosis by inhibiting thrombin activity, platelet activation, and finally macrophage-mediated plaque inflammation.

Prothrombotic SERPINC1 gene polymorphism may affect heparin sensitivity among different ethnicities of Chinese patients receiving heart surgery.

The purpose of this study was to investigate a possible correlation between single-nucleotide polymorphisms (SNPs) of the antithrombin (gene, SERPINC1, and perioperative sensitivity to heparin in patients receiving heart surgery. The SERPINC1 genotype and allele frequency, coagulation parameters 24 hours before and after surgery, and clinical findings were compared among 3 ethnic groups, Han, Uighur, and Kazakh, patientswho received heart surgery. In Han patients, longer coagulation time as well as higher heparin and protamine dosage was observed. SERPINC1 gene sequencing identified 2 mutations in exon 5, g.981A>G (rs5877) and g.1011A>G (rs5878). The minor allele frequency of allele (A>G) for rs5877 and rs5878 was higher in the Han patients and was significantly different among the ethnic groups (P = .004 and P = .006, respectively). The increased SERPINC1 SNP frequency among Han patients receiving heart surgery might contribute to the differences in their perioperative sensitivity to heparin.

Liver-directed gene therapy corrects cardiovascular lesions in feline mucopolysaccharidosis type I.

Patients with mucopolysaccharidosis type I (MPS I), a genetic deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), exhibit accumulation of glycosaminoglycans in tissues, with resulting diverse clinical manifestations including neurological, ocular, skeletal, and cardiac disease. MPS I is currently treated with hematopoietic stem cell transplantation or weekly enzyme infusions, but these therapies have significant drawbacks for patient safety and quality of life and do not effectively address some of the most critical clinical sequelae, such as life-threatening cardiac valve involvement. Using the naturally occurring feline model of MPS I, we tested liver-directed gene therapy as a means of achieving long-term systemic IDUA reconstitution. We treated four MPS I cats at 3-5 mo of age with an adeno-associated virus serotype 8 vector expressing feline IDUA from a liver-specific promoter. We observed sustained serum enzyme activity for 6 mo at ∼ 30% of normal levels in one animal, and in excess of normal levels in three animals. Remarkably, treated animals not only demonstrated reductions in glycosaminoglycan storage in most tissues, but most also exhibited complete resolution of aortic valve lesions, an effect that has not been previously observed in this animal model or in MPS I patients treated with current therapies. These data point to clinically meaningful benefits of the robust enzyme expression achieved with hepatic gene transfer that extend beyond the economic and quality of life advantages over lifelong enzyme infusions.

The complete N-terminal extension of heparin cofactor II is required for maximal effectiveness as a thrombin exosite 1 ligand.

BACKGROUND: Heparin cofactor II (HCII) is a circulating protease inhibitor, one which contains an N-terminal acidic extension (HCII 1-75) unique within the serpin superfamily. Deletion of HCII 1-75 greatly reduces the ability of glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, and abrogates HCII binding to thrombin exosite 1. While a minor portion of HCII 1-75 can be visualized in a crystallized HCII-thrombin S195A complex, the role of the rest of the extension is not well understood and the affinity of the HCII 1-75 interaction has not been quantitatively characterized. To address these issues, we expressed HCII 1-75 as a small, N-terminally hexahistidine-tagged polypeptide in E. coli. RESULTS: Immobilized purified HCII 1-75 bound active α-thrombin and active-site inhibited FPR-ck- or S195A-thrombin, but not exosite-1-disrupted γT-thrombin, in microtiter plate assays. Biotinylated HCII 1-75 immobilized on streptavidin chips bound α-thrombin and FPR-ck-thrombin with similar KD values of 330-340 nM. HCII 1-75 competed thrombin binding to chip-immobilized HCII 1-75 more effectively than HCII 54-75 but less effectively than the C-terminal dodecapeptide of hirudin (mean Ki values of 2.6, 8.5, and 0.29 µM, respectively). This superiority over HCII 54-75 was also demonstrated in plasma clotting assays and in competing the heparin-catalysed inhibition of thrombin by plasma-derived HCII; HCII 1-53 had no effect in either assay. Molecular modelling of HCII 1-75 correctly predicted those portions of the acidic extension that had been previously visualized in crystal structures, and suggested that an α-helix found between residues 26 and 36 stabilizes one found between residues 61-67. The latter region has been previously shown by deletion mutagenesis and crystallography to play a crucial role in the binding of HCII to thrombin exosite 1. CONCLUSIONS: Assuming that the KD value for HCII 1-75 of 330-340 nM faithfully predicts that of this region in intact HCII, and that 1-75 binding to exosite 1 is GAG-dependent, our results support a model in which thrombin first binds to GAGs, followed by HCII addition to the ternary complex and release of HCII 1-75 for exosite 1 binding and serpin mechanism inhibition. They further suggest that, in isolated or transferred form, the entire HCII 1-75 region is required to ensure maximal binding of thrombin exosite 1.

Heparin cofactor II, a serine protease inhibitor, promotes angiogenesis via activation of the AMP-activated protein kinase-endothelial nitric-oxide synthase signaling pathway.

We previously clarified that heparin cofactor II (HCII), a serine proteinase inhibitor, exerts various protective actions on cardiovascular diseases in both experimental and clinical studies. In the present study, we aimed to clarify whether HCII participates in the regulation of angiogenesis. Male heterozygous HCII-deficient (HCII(+/-)) mice and male littermate wild-type (HCII(+/+)) mice at the age of 12-16 weeks were subjected to unilateral hindlimb ligation surgery. Laser speckle blood flow analysis showed that blood flow recovery in response to hindlimb ischemia was delayed in HCII(+/-) mice compared with that in HCII(+/+) mice. Capillary number, arteriole number, and endothelial nitric-oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and liver kinase B1 (LKB1) phosphorylation in ischemic muscles were decreased in HCII(+/-) mice. Human purified HCII (h-HCII) administration almost restored blood flow recovery, capillary density, and arteriole number as well as phosphorylation levels of eNOS, AMPK, and LKB1 in ischemic muscles of HCII(+/-) mice. Although treatment with h-HCII increased phosphorylation levels of eNOS, AMPK, and LKB1 in human aortic endothelial cells (HAECs), the h-HCII-induced eNOS phosphorylation was abolished by compound C, an AMPK inhibitor, and by AMPK siRNA. In a similar fashion, tube formation, proliferation, and migration of HAECs were also promoted by h-HCII treatment and were abrogated by pretreatment with compound C. HCII potentiates the activation of vascular endothelial cells and the promotion of angiogenesis in response to hindlimb ischemia via an AMPK-eNOS signaling pathway. These findings suggest that HCII is a novel therapeutic target for treatment of patients with peripheral circulation insufficiency.

Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli.

Irreversible inactivation of alpha-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (K(D)) of 6+/-1 and 7+/-1 microM, respectively, approximately 6-fold tighter than plasma HCII, with K(D) 40+/-4 microM. Binding of recombinant and deglycosylated HCII to DS, both with K(D) 4+/-1 microM, was approximately 4-fold tighter than for plasma HCII, with K(D) 15+/-4 microM. Recombinant HCII, lacking N-glycosylation and tyrosine sulfation, inactivated alpha-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn(169)N-glycan with the HCII heparin-binding site.

Antithrombin and heparin cofactor II-mediated inactivation of alpha-thrombin by a synthetic, sulfated mannogalactan.

INTRODUCTION: A mannogalactan from Pleurotus ostreatoroseus (MgPr) was chemically sulfated to give MgPr-S1, which was evaluated for its anticoagulant and antithrombotic activities, bleeding tendency, and platelet aggregation. MATERIALS AND METHODS: MgPr-S1 was partially characterized by HPSEC-MALLS, methylation analysis, and 13C NMR spectroscopy. Its anticoagulant activity was determined by assays of aPTT, TT, alpha-thrombin and factor Xa residual activity, heparin cofactor II (HCII)-, or antithrombin (AT)-mediated inhibition. The antithrombotic effect was evaluated in rats using a venous thrombosis model and the bleeding tendency was also tested in vivo. Platelet aggregation was investigated by an adaptation of the method of Born [1]. RESULTS: The hydroxyl groups of beta-D-Manp units and OH-2 and OH-4 of the (1-->6)-linked alpha-D-Galp units were preferentially substituted. The anticoagulant activity of MgPr-S1 was mainly by thrombin inhibition with antithrombin and HCII, and had an effect on platelet aggregation induced by ADP and alpha-thrombin. It almost completely inhibited thrombus formation in vivo at a dose of 6 mg/kg and heparin inhibited thrombus formation at a dose of 0.200 mg/kg. CONCLUSIONS: These results suggested that the chemically sulfated mannogalactan could act as an alternative to heparin as anticoagulant.

Evaluation of heparin cofactor II-thrombin complex as a biomarker on blood spots from mucopolysaccharidosis I, IIIA and IIIB mice.

Mucopolysaccharide (MPS) diseases are lysosomal storage disorders caused by deficiencies of enzymes catabolising glycosaminoglycans (GAGs). Abnormal GAG accumulation leads to symptoms including severe progressive neurological decline, skeletal deformities, organomegally, respiratory compromise and premature death. Treatment is available for some MPS diseases; enzyme replacement therapy for MPS I, II and VI, and haematopoietic stem cell transplantation for MPS I, VI and VII. These treatments are reliant on early diagnosis of the disease and accurate monitoring of treatment outcomes. Blood enzyme levels and total urinary GAGs are commonly used biomarkers in diagnosis of MPS but are not good measures of treatment outcome. Serum heparin cofactor II-thrombin complex (HCII-T), which is a GAG regulated serpin-protease complex, has recently been identified as a promising biomarker for MPS diseases. Here we present an assessment of the HCII-T biomarker in mouse models of MPS I, IIIA and IIIB, which suggests that HCII-T is a reliable marker for MPS I when measured in serum or dried blood spots stored for over a year at 4 degrees C, but that murine MPS IIIA and IIIB cannot be reliably detected using this biomarker. We also show that HCII-T formation in vivo is dependent on the presence of excess intravenous dermatan sulphate (DS), whilst intravenous heparan sulphate (HS), does not promote complex formation effectively. This suggests that HCII-T will prove effective as a biomarker for MPS I, II, VI and VII diseases, storing dermatan sulphate but may not be as appropriate for MPS III, storing heparan sulphate. With careful sample preparation, HCII-T ELISA could prove to be a useful biomarker for both newborn screening and measurement of treatment outcomes in selected MPS diseases.

Effects of antithrombin and heparin cofactor II levels on anticoagulation with Intimatan.

Heparin is the current mainstay drug for anticoagulation during cardiac surgery, but it requires normal levels of antithrombin (AT) for optimal anticoagulation. Heparin anticoagulation may be less effective in cardiac surgical patients because of decreased AT levels due to the prolonged heparin therapy. Therefore, other anticoagulants that would work well in AT deficient patients may be more desirable. One such agent currently being evaluated is Intimatan, which catalyzes heparin cofactor II (HCII) dependent inhibition of thrombin. In the current in vitro study we examined the effects of Intimatan (20 microg/ml), heparin (0.25 U/ml), or both drugs in combination on thrombin generation in plasma with decreasing levels of AT, HCII or both cofactors, using a novel method based on the continuous measurement of thrombin generation. For the study, we collected blood samples from healthy volunteers, isolated platelet poor plasma by centrifugation and mixed it with AT, HCII, or AT-HCII deficient plasma samples to achieve different levels of AT, HCII and AT-HCII. Thrombin generation was inhibited equally well with heparin or Intimatan when the level of their respective cofactors was within the normal range. With decreasing levels of AT or HCII, heparin and Intimatan became less effective in thrombin inhibition, respectively. With the absence of both cofactors, neither agent alone or in combination had any effect on thrombin generation. We conclude that Intimatan may be an effective adjunct to heparin therapy under low AT conditions.

Plasma levels of heparin cofactor II (HCII) and thrombin-HCII complex in patients with disseminated intravascular coagulation.

Plasma levels of heparin cofactor II (HCII), thrombin-HCII complex (THC), antithrombin (AT), and thrombin-AT complex (TAT) were evaluated in patients with disseminated intravascular coagulation (DIC) associated with several underlying diseases. Plasma levels of AT were significantly reduced in almost all underlying diseases associated with DIC, but the plasma levels of HCII and HCII/AT ratio were significantly reduced only in patients with infections. While the plasma level of TAT was significantly increased in patients with all underlying diseases associated with DIC, the increase of THC was not significant. Plasma levels of AT were significantly reduced in DIC and pre-DIC associated with almost all underlying diseases, but those of HCII were significantly reduced only in DIC and pre-DIC patients with inflammatory diseases. The plasma levels of TAT were significantly increased in DIC, pre-DIC, and non-DIC patients with all underlying diseases, and those of THC were significantly increased in DIC and pre-DIC patients with inflammatory diseases. The plasma levels of THC were not significantly increased in non-DIC patients of any disease group. The decrease of AT may be caused by thrombin generation or inflammatory reaction that occurs in DIC associated with underlying diseases, while the decrease of HCII might be caused by both thrombin generation and inflammatory reaction. Finally, AT inhibits thrombin more strongly than HCII in several underlying diseases associated with DIC except for inflammatory diseases. In inflammatory diseases, HCII might play an important role in preventing the onset of DIC.

Contribution of basic residues of the A helix of heparin cofactor II to heparin- or dermatan sulfate-mediated thrombin inhibition.

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated 1000-fold by heparin or dermatan sulfate. To investigate the contribution of basic residues of the A helix of HCII to this activation, we constructed amino acid substitutions (K101Q, R103L, and R106L) by site-directed mutagenesis. K101Q greatly reduced heparin cofactor activity and required a more than 10-fold higher concentration of dermatan sulfate to accelerate thrombin inhibition compared with wild-type recombinant HCII. Thrombin inhibition by R106L was not significantly stimulated by dermatan sulfate. These results provide evidence that basic residues of the A helix of HCII (Lys(101) and Arg(106)) are necessary for heparin- or dermatan sulfate-accelerated thrombin inhibition.

The procoagulant state of the VX-2 tumor in rabbit lung in vivo--relative accumulation of fibrinogen, prothrombin, plasminogen, antithrombin and heparin cofactor II within the tumor.

During their growth, many malignant solid tumors elicit a hemostatic response in the host. In this report, the fluxes of various rabbit plasma hemostatic proteins into pulmonary VX-2 tumors have been measured in vivo. VX-2 cells were contained within a small rabbit plasma clot which was injected intravenously into rabbits. At 28 d, each rabbit was injected intravenously with two radiolabeled (131I, 125I) proteins selected from fibrinogen, prothrombin, antithrombin-alpha, heparin cofactor II, or plasminogen-I or -II. After allowing the labeled proteins to circulate for 10-200 min, each rabbit was perfused with Krebs-Henseleit solution and the lungs excised. Solid tumors were isolated, weighed and measured for radioactivity content. A mean of 14 tumors/pair of lungs with a mean tumor weight of 0.3 g was obtained. Radioactivity per g of tumor was related to radioactivity/ml of blood at exsanguination for each protein at each time interval. Maximum flux rates were calculated (as pmol/g of tumor/min): Fibrinogen, 65.0; prothrombin, 53.0; antithrombin-alpha, 24.1; HCII, 17.2; plasminogen-II, 5.0; and plasminogen-I, 3.2. Using immunocytochemical staining, fibrin(ogen) was distributed heterogeneously within the VX-2 tumor, appearing largely in the perinodular region and in the necrotic core. From the net fluxes of these proteins, the profile of chronic hemostasis associated with the VX-2 tumor was shown to differ markedly from the hemostatic response that occurs after an acute vascular injury.

Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II.

Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycan-binding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant D, with changes at Arg(184), Lys(185), Arg(189), Arg(192), Arg(193), demonstrated a approximately 130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates gamma-thrombin at a 65-fold slower rate than alpha-thrombin, (b) hirudin-(54-65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation approximately 100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.