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The transformation of vascular smooth muscle cells [VSMC] into foam cells leading to increased plaque size and decreased stability is a key, yet understudied step in atherogenesis. We reported that Interleukin-19 (IL-19), a novel, anti-inflammatory cytokine, attenuates atherosclerosis by anti-inflammatory effects on VSMC. In this work we report that IL-19 induces expression of miR133a, a muscle-specific miRNA, in VSMC. Although previously unreported, we report that miR133a can target and reduce mRNA abundance, mRNA stability, and protein expression of Low Density Lipoprotein Receptor Adaptor Protein 1, (LDLRAP1), an adaptor protein which functions to internalize the LDL receptor. Mutations in this gene lead to LDL receptor malfunction and cause the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans. Herein we show that IL-19 reduces lipid accumulation in VSMC, and LDLRAP1 expression and oxLDL uptake in a miR133a-dependent mechanism. We show that LDLRAP1 is expressed in plaque and neointimal VSMC of mouse and human injured arteries. Transfection of miR133a and LDLRAP1 siRNA into VSMC reduces their proliferation and uptake of oxLDL. miR133a is significantly increased in plasma from hyperlipidemic compared with normolipidemic patients. Expression of miR133a in IL-19 stimulated VSMC represents a previously unrecognized link between vascular lipid metabolism and inflammation, and may represent a therapeutic opportunity to combat vascular inflammatory diseases.

Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth factor (CTGF/CCN2) on fibroblasts. Since tissue factor (TF) is expressed on these cells, we hypothesized that they may assemble the prothrombinase complex leading to CTGF/CCN2 upregulation. In addition, the effect of thrombospondin-1 (TSP1) on this reaction was evaluated. Human foreskin fibroblasts were incubated with purified factor VII (FVII), factor X (FX), factor V (FV), prothrombin and calcium in the presence and absence of TSP1. Generation of FXa and of thrombin were assessed using chromogenic substrates. SMAD pathway phosphorylation was detected via Western-blot analysis. Pre-incubation of fibroblasts with FVII led to its auto-activation by cell-surface expressed TF, which in turn in the presence of FX, FVa, prothrombin and calcium led to FXa (9.7 ± 0.8 nM) and thrombin (7.9 ± 0.04 U/mL × 10–3) generation. Addition of TSP1 significantly enhanced thrombin (23.3 ± 0.7 U/mL × 10–3) but not FXa (8.5 ± 0.6 nM) generation. FXa and thrombin generation leads to upregulation of CTGF/CCN2. TSP1 alone upregulated CTGF/CCN2, an effect mediated via activation of transforming growth factor beta (TGFβ) as shown by phosphorylation of the SMAD pathway, an event blunted by using a TGFβ receptor I inhibitor (TGFβRI). FXa- and thrombin-induced upregulation of CTGF/CCN2 was not blocked by TGFβRI. In summary, assembly of the prothrombinase complex occurs on fibroblast's surface leading to serine proteases generation, an event enhanced by TSP1 and associated with CTGF/CCN2 upregulation. These mechanisms may play an important role in human diseases associated with fibrosis.

Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morphogenetic proteins (BMPs), which is costly, and may not replicate normal bone healing. The limited in vivo biologic activity of BMPs requires the investigation of growth factors that may enhance this activity. In this study, we utilized the C3H10T1/2 murine mesenchymal stem cell line to test the hypotheses that osteoactivin (OA) has comparable osteoinductive effects to bone morphogenetic protein-2 (BMP-2), and that sustained administration of either growth factor would result in increased osteoblastic differentiation as compared to bolus administration. Sustained release biodegradable hydrogels were designed, and C3H10T1/2 cells were grown on hydrogels loaded with BMP-2 or OA. Controls were grown on unloaded hydrogels, and positive controls were exposed to bolus growth factor administration. Cells were harvested at several time points to assess osteoblastic differentiation. Alkaline phosphatase (ALP) staining and activity, and gene expression of ALP and osteocalcin were assessed. Treatment with OA or BMP-2 resulted in comparable effects on osteoblastic marker expression. However, cells grown on hydrogels demonstrated osteoblastic differentiation that was not as robust as cells treated with bolus administration. This study shows that OA has comparable effects to BMP-2 on osteoblastic differentiation using both bolus administration and continuous release, and that bolus administration of OA has a more profound effect than administration using hydrogels for sustained release. This study will lead to a better understanding of appropriate delivery methods of osteogenic growth factors like OA for repair of fractures and segmental bone defects.

Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA die prematurely, in part due to associated cardiovascular disease. Even though the mechanisms by which premature atherosclerosis develops in RA is unknown, chronic inflammation may play a major role. This review connects current knowledge of the pathophysiology of RA with data available in the literature related to thrombospondin-1 (TSP1), transforming growth factor beta (TGFbeta and connective tissue growth factor (CTGF) and their relationship with cardiovascular disease in RA. The TSP1/TGFbeta/CTGF axis may contribute in the pro-inflammatory and pro-atherogenic state in patients affected with RA. In fact, increased TSP1 plasma levels are found in patients of RA. TGFbeta is activated by TSP1 through a non-enzymatic mechanism and is constitutively overexpressed by synovial fibroblasts from RA patients. Activation of TGFbeta pathway in synovial fibroblasts and other cells including neutrophils leads to downstream upregulation of CTGF. Overexpression of CTGF is associated with angiogenesis, fibrosis, atherosclerotic blood vessels and erosive arthritis lesions. Recent RA therapies emphasize the need for aggressive control of the activity of the disease to prevent premature atherosclerosis in RA patients. The complexity and heterogeneity of RA as judged by response to a wide spectrum of treatments mandates the elucidation of unknown pro-inflammatory pathways playing a major role in this disease. The TSP1/TGFbeta/CTFG axis represents one of these pro-inflammatory pathways that may result in the development of promising therapeutic strategies to prevent chronic inflammation and thus premature atherosclerosis in RA.

Thrombospondin-1 (TSP1/THBS1) plays a major role in the pathophysiology of rheumatoid arthritis (RA); however, its interface with the cytokine network involved in RA has not been delineated. Correlations were performed between plasma levels of TSP1 and selected cytokines from blood samples collected from 20 patients affected by RA and 13 healthy donors (control). Plasma levels of TSP1 and tissue growth factor beta (TGFbeta) were determined by standard enzyme-linked immunosorbent assay, and cytokines were measured by protein profiling rolling-circle amplification (RCA). TSP1 circulating levels in plasma were found significantly increased in the RA patients when compared with control individuals (P = 0.039). The plasma levels of TGFbeta were also increased in the RA patients, which indicates a statistical trend. Cytokine levels of interleukin (IL)-4, IL-5, IL-12, chemokine CXC 10 (CXCL10/IP10), and chemokine CC 4 (CCL4)/MIP1beta were significantly increased in the RA patients when compared with the control group. In summary, this study demonstrates increased plasma levels of TSP1, which correlated with increased levels of proinflammatory cytokines in plasma of RA patients. More detailed research is required to explore the cytokine imprint yielded by this study and its interface with TSP1 and TGFbeta.

Objective To compare inflammatory peripheral arthritis in wild-type and high molecular weight kininogen (HK)–deficient rats, both on the genetically susceptible Lewis background. Methods By backcrossing Brown-Norway HK-deficient rats with Lewis rats for 6 generations, 2 new strains were produced, wild-type F6 and HK-deficient F6, each with a 98.5% Lewis genome. Inflammatory arthritis was induced by intraperitoneal injection of peptidoglycan–polysaccharide (PG-PS), and the clinical, histopathologic, and biochemical responses were compared in both strains. Results Eighteen days after PG-PS injection, rats with normal concentrations of HK showed weight loss and marked increase in hind ankle diameter with severe synovial inflammation and cartilage abnormalities. In contrast, HK-deficient rats showed no weight loss (P < 0.05), no increase in hind ankle diameter (P < 0.05), and an absence of inflammatory changes (P < 0.05), as measured by the histologic and morphometric Mankin grading system for synovial and cartilage injury. Conclusion Plasma HK is a key mediator of acute and chronic inflammatory arthritis in genetically susceptible Lewis rats.

Introduction Thrombospondin 1 (TSP1) has the ability to bind to HL-60 cells and to reversibly inhibit human neutrophil elastase (HNE). Human factor V (FV) can be cleaved by HNE thereby providing FV with cofactor activity (FVa HNE ). Experiments were performed to evaluate the ability of HNE expressed on the surface of HL-60 cells to generate FVa HNE to support thrombin generation, and to determine the effect of TSP1 on this reaction. Results Western blot analysis showed TSP1 forming a complex with FVa HNE within a region corresponding to the heavy chain of FV. Enzymatic reactions were performed to determine the role of TSP1–HNE–FVa HNE on the surface of HL-60 cells, namely the assembly of the prothrombinase complex. Thrombin generation was measured by the chromogenic substrate S2238. Exposure of factor V to HL-60 cells prior to the addition of prothrombin and activated factor X provided FV with cofactor activity. HL-60 cells were found capable of synthesizing factor V with cofactor activity, but HL-60 cells failed to synthesize and/or to provide factor X with enzymatic activity. The ability of HL-60 cells to synthesize FV and TSP1 was demonstrated. The addition of exogenous TSP1 enhanced both the rate and amount of thrombin generated on the HL-60 cell surface. Conclusion Despite the ability of TSP1 to reversibly inhibit HNE in a purified system, TSP1 expression favors the reactions leading to thrombin generation on the HL-60 cell surface. These observations are relevant to clinical conditions where there is a prothrombotic state such as malignant tumors.

We reported that high-molecular weight kininogen is proangiogenic by releasing bradykinin and that a monoclonal antibody to high-molecular weight kininogen, C11C1, blocked its binding to endothelial cells. We now test if this antibody can prevent arthritis and systemic inflammation in a Lewis rat model. We studied 32 animals for 16 days. Group I (negative control) received saline intraperitoneally. Group II (disease-treated) received peptidoglycan-polysaccharide simultaneously with C11C1. Group III (disease-untreated) received peptidoglycan-polysaccharide simultaneously with isotype-matched mouse IgG. Group IV (disease-free-treated) and group V (disease-free isotype-treated) received saline and C11C1 or mouse IgG. Analysis of joint diameter changes showed a decrease in the C11C1 disease-treated group compared to the disease-untreated group. The hind paw inflammatory score showed a decrease in the intensity and extent of inflammation between the disease-untreated and the C11C1 disease-treated group. Prekallikrein, high-molecular weight kininogen, factor XI, and factor XII were decreased in the disease-untreated group compared to the C11C1 disease-treated group. T-kininogen was increased in the disease-untreated group when compared with the C11C1 disease-treated group. Disease-free groups IV and V did not show any sign of inflammation at any time. This study shows that monoclonal antibody C11C1 attenuates plasma kallikrein-kinin system activation, local and systemic inflammation, indicating therapeutic potential in reactive arthritis.

Robert Kaplan, Hematology Fellow, Department ofMedicine and Sol Sherry Thrombosis Research Center,Temple University School of Medicine: The patient is a64-year-old Caucasian female with a history of hyper-tension and diabetes mellitus who presented with a four-month history of weight loss and fatigue. Laboratoryevaluation revealed a pancytopenia initially suspected tobe related to her multiple medications. Despite changesin her therapeutic regimen, there was no significant im-provement in her blood cell counts. A subsequent bonemarrow aspirate revealed atypical promyelocytes withabundant Auer rods accounting for 35% of the marrowcells. Cytogenetic studies detected the t(15,17)(q

The binding of high-molecular-weight kininogen (HK) to neutrophils (polymorphonuclear leukocytes, PMN) is required for the stimulation of aggregation and degranulation by human plasma kallikrein as well as the displacement of fibrinogen from this cell surface. The putative receptor for HK is the leukocyte integrin αMβ2, and domains 3 (D3) and 5 (D5) of HK form its binding site. To further map the binding sites on HK for PMN, we used D3 recombinant exon products and designed peptides from D3 and D5. In D3, a heptapeptide, Leu271-Ala277, from exon 7 product, and a peptide, Cys333-Cys352, from exon 9 product can inhibit binding of kininogen to PMN. Two contiguous peptides from D5 in the histidine-glycine-rich region, Gly442-Lys458and Phe459-Lys478, each inhibit the binding of HK to PMN. This study has thus delineated three noncontiguous surface-oriented sequences on HK, which together comprise all or most of the binding site for human PMN.

We and others have shown that both high and low molecular mass kininogens are able to inhibit the thrombin-induced aggregation of gel-filtered platelets, indicating that the locus for inhibition resides in the heavy chain. The inhibitory site is present in domain 3, confined to the C-terminal portion of the region encoded by exon 7 (K270-G292), and the minimal effective sequence is a heptapeptide (L271-A277; Kunapuli et al, J Biol Chem 271:11228, 1996). Kininogens inhibit thrombin binding to platelets and thus inhibit thrombin-induced aggregation. The molecular mechanism by which kininogens inhibit thrombin-induced aggregation of platelets is unknown. Thrombin has previously been shown to bind to two receptors on the platelet surface, glycoprotein (GP) Ib-IX-V complex and the hepta-spanning transmembrane receptor coupled to G protein(s). We now show that, unlike its effect on normal platelets, kininogen (2 μmol/L) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GP Ib-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex and restricts access by thrombin to this receptor. We further show that both recombinant K270-G292 polypeptide and the synthetic peptide L271-A277 derived from high molecular mass kininogen lower thrombin binding to platelets in a manner similar to monoclonal antibodies to or ligands (von Willebrand factor and echicetin) of GP Ib-IX. The anti–GP Ib-IX-V complex antibodies, TM-60 and SZ 2, can inhibit 125I-high molecular mass kininogen binding to platelets. Conversely, kininogen could block the binding of biotinylated TM-60 or of 125I-SZ 2. Kininogen inhibited the binding of biotinylated thrombin bound to a mouse fibroblast cell line transfected with the GP Ib-IX-V complex. These results indicated that kininogen binds to the GP Ib-IX-V complex modulating thrombin binding to platelets and the consequent platelet aggregation. Kininogen can thus serve as an important regulator of the early stages of platelet stimulation by thrombin.

We and others have shown that both high and low molecular mass kininogens are able to inhibit the thrombin-induced aggregation of gel-filtered platelets, indicating that the locus for inhibition resides in the heavy chain. The inhibitory site is present in domain 3, confined to the C-terminal portion of the region encoded by exon 7 (K270-G292), and the minimal effective sequence is a heptapeptide (L271-A277; Kunapuli et al, J Biol Chem 271:11228, 1996). Kininogens inhibit thrombin binding to platelets and thus inhibit thrombin-induced aggregation. The molecular mechanism by which kininogens inhibit thrombin-induced aggregation of platelets is unknown. Thrombin has previously been shown to bind to two receptors on the platelet surface, glycoprotein (GP) Ib-IX-V complex and the hepta-spanning transmembrane receptor coupled to G protein(s). We now show that, unlike its effect on normal platelets, kininogen (2 μmol/L) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GP Ib-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex and restricts access by thrombin to this receptor. We further show that both recombinant K270-G292 polypeptide and the synthetic peptide L271-A277 derived from high molecular mass kininogen lower thrombin binding to platelets in a manner similar to monoclonal antibodies to or ligands (von Willebrand factor and echicetin) of GP Ib-IX. The anti–GP Ib-IX-V complex antibodies, TM-60 and SZ 2, can inhibit 125I-high molecular mass kininogen binding to platelets. Conversely, kininogen could block the binding of biotinylated TM-60 or of 125I-SZ 2. Kininogen inhibited the binding of biotinylated thrombin bound to a mouse fibroblast cell line transfected with the GP Ib-IX-V complex. These results indicated that kininogen binds to the GP Ib-IX-V complex modulating thrombin binding to platelets and the consequent platelet aggregation. Kininogen can thus serve as an important regulator of the early stages of platelet stimulation by thrombin.

Administration of the thienopyridine P2Y12 receptor antagonist, clopidogrel, increased the erosive arthritis induced by peptidoglycan polysaccharide (PG-PS) in rats or by injection of the arthritogenic K/BxN serum in mice. To determine if the detrimental effects are caused exclusively by clopidogrel, we evaluated prasugrel, a third-generation thienopyridine pro-drug, that contrary to clopidogrel is mostly metabolized into its active metabolite in the intestine. Prasugrel effects were examined on the PG-PS-induced arthritis rat model. Erosive arthritis was induced in Lewis rats followed by treatment with prasugrel for 21 days. Prasugrel treated arthritic animals showed a significant increase in the inflammatory response, compared with untreated arthritic rats, in terms of augmented macroscopic joint diameter associated with significant signs of inflammation, histomorphometric measurements of the hind joints and elevated platelet number. Moreover, fibrosis at the pannus, assessed by immunofluorescence of connective tissue growth factor, was increased in arthritic rats treated with prasugrel. In addition to the arthritic manifestations, hepatomegaly, liver granulomas and giant cell formation were observed after PG-PS induction and even more after prasugrel exposure. Cytokine plasma levels of IL-1 beta, IL-6, MIP1 alpha, MCP1, IL-17 and RANTES were increased in arthritis-induced animals. IL-10 plasma levels were significantly decreased in animals treated with prasugrel. Overall, prasugrel enhances inflammation in joints and liver of this animal model. Since prasugrel metabolites inhibit neutrophil function ex-vivo and the effects of both clopidogrel and prasugrel metabolites on platelets are identical, we conclude that the thienopyridines metabolites might exert non-platelet effects on other immune cells to aggravate inflammation.

Increased life expectancy and the need for long-term antiretroviral therapy have brought new challenges to the clinical management of HIV-infected individuals. The prevalence of osteoporosis and fractures is increased in HIV-infected patients; thus optimal strategies for risk management and treatment in this group of patients need to be defined. Prevention of bone loss is an important component of HIV care as the HIV population grows older. Understanding the mechanisms by which HIV infection affects bone biology leading to osteoporosis is crucial to delineate potential adjuvant treatments. This review focuses on HIV-induced osteoporosis within the context of microRNAs (miRNAs) by reviewing first basic concepts of bone biology as well as current knowledge of the role of miRNAs in bone development. Evidence that HIV-associated osteoporosis is in part independent of therapies employed to treat HIV (HAART) is supported by cross-sectional and longitudinal studies and is the focus of this review.

Severe progressive failure of multiple organ systems has emerged during the past three decades as a common cause of death among patients in intensive care units. Sepsis is now better defined as systemic inflammatory response syndrome (SIRS), but its mortality rate has not changed and it continues to be a major health problem. Endotoxin interacts with plasma proteins and contributes to the pathophysiology of SIRS. Information is limited on the effect of endotoxin on human coagulation and fibrinolytic proteins in vivo, as well as on the cell response involved in the cytokine cascade. For this reason we performed quantitative assays to establish the sequence of events that occurs in vivo in the regulation of the contact and fibrinolytic pathways as well as in the cytokine cascade as a response to a single dose administration of endotoxin to normal non-smoking human volunteers.

The ability of kininogens to modulate thrombin-induced aggregation of human platelets has been assigned to domain 3 (D3) in the common heavy chain coded for by exons 7, 8, and 9 of kininogen gene. We expressed each of the exons 7, 8, and 9, and various combinations as glutathione S-transferase fusion proteins in Escherichia coli. Each of the exon products 7 (Lys236-Gln292), 9 (Val293-Gly328), and 8 (Gln329-Met357), and their combinations were evaluated for the ability to inhibit thrombin induced platelet aggregation. Only products containing exon 7 inhibited platelet aggregation induced by thrombin with an IC50 of > 20 microM. A deletion mutant of exon 7 product, polypeptide 7A product (Lys236-Lys270) did not block thrombin-induced platelet aggregation, while 7B product (Thr255-Gln292) and 7C product (Leu271-Gln292) inhibited aggregation. These findings indicated that the inhibitory activity is localized to residues Leu271-Gln292. Peptides Phe279-Ile283 and Phe281-Gln292 did not block thrombin, and Asn275-Phe279 had only minimal inhibitory activity. A heptapeptide Leu271-Ala277 inhibited thrombin-induced aggregation of platelets with an IC50 of 65 microM. The effect is specific for the activation of platelets by thrombin but not ADP or collagen. No evidence for a thrombin-kininogen complex was found, and neither HK nor its derivatives directly inhibited thrombin activity. Knowledge of the critical sequence of kininogen should allow design of compounds that can modulate thrombin activation of platelets.

This Report from the Field documents a series of interventions developed by Temple University Health System and School of Medicine through participation in the RWJF initiative entitled Hablamos Juntos. The report delineates outcomes to date demonstrating that these interventions have met the challenge of improving patient provider communication for Latinos.

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation.

Evidence has accumulated over the past three decades implicating plasma kininogens in numerous inflammatory processes. Delineation of the detailed biochemistry and, more recently, the molecular biology of the human kininogens has resulted in a deeper understanding of the structure-function correlations of the human kininogens. Studies of alterations of human kininogens in disease states have yielded information about the mechanisms of their involvement in inflammatory states. Here, Raul DeLa Cadena and Robert Colman summarize kirinogen function in relation to structure and diagnostic and therapeutic potential.

Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. Results: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. Conclusion: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues. J. Cell. Physiol. 211: 504–512, 2007. © 2007 Wiley-Liss, Inc.

Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with leukocyte adhesion to and extravasation through vascular endothelium into synovial tissue. Recent evidence indicates that the thrombospondin 1 gene is up-regulated in patients with RA. We have identified a region within the TSP-1 type 3 repeats that inhibits human neutrophil elastase (HNE) and binds to human neutrophils. The present study was undertaken to investigate the therapeutic benefit of this TSP-1-derived peptide sequence and its effect on connective tissue growth factor (CTGF), a protein involved in fibrotic disorders and in neovascularization, which is a hallmark of RA. Methods CTGF gene and protein expression, as well as protein levels of CTGF in the synovium, after treatment with the TSP-1-derived peptide were studied in the peptidoglycan-polysaccharide animal model of erosive arthritis. Results Peptide treatment prevented joint infiltration and inflammation and was associated with reduced circulating antigen levels of HNE and TSP-1. Additionally, CTGF was up-regulated in this experimental model of RA. Treatment with the TSP-1-derived peptide was associated with down-regulation of the message and protein levels of CTGF. Immunofluorescence studies showed that the mean area fraction of CTGF immunoreactivity in the peptide-treated group of animals was significantly less than that in the untreated group. Conclusion These results document a role for TSP-1 in regulating CTGF gene and protein expression in synovial tissue, suggesting a link with the disease course in this model of RA. This TSP-1-derived synthetic peptide may represent an important template for drug development in RA and other inflammatory conditions associated with neutrophil activation.

Introduction: Diabetic retinopathy (DR) is a severe ocular complication of type 2 diabetes (T2DM). Our laboratory has been studying the role of a pro-inflammatory axis comprised by TSP1-TGFb and CTGF in T2DM. The axis is differentially expressed in patients with T2DM and non-proliferative DR (NPDR) when compared with patients with T2DM and proliferative DR (PDR). The differential expression of such axis is accompanied by interesting cytokine profiles as well. The purpose of this study was to evaluate potential microRNAs associated with the above changes in order to understand better the pathophysiology of T2DM. Recent studies indicate that miRNAs transported to exosomes or HDL can be transferred in an active form to recipients cells potentially involving them in cell-to-cell communication. Methods: Institutional approved IRB and informed consent allowed this prospective study to recruit a total of 40 individuals afflicted by T2DM (n=20 NPDR and n=20 PDR) from the Ophthalmology Department Diabetes Clinic at Temple Hospital, normal controls (n=20) were recruited from the Sol Sherry Thrombosis Research Center at Temple. Western-blotting technique was utilized to document the presence of fragments from CTGF in plasma, which have been documented in vitreous fluid to be angiogenic. Multiplexed protein profiling on microarrays by rolling-circle amplification was employed to determine IL-4 and MIP-1b. Commercially available ELISA was the method to determine the plasma concentration of TSP1, TGFb and CTGF. Total RNA was extracted from human plasma using the miRCURY TM RNA isolation kit by Exiqon at their facility in Denmark. Results and Discussion: TSP1, TGFb, CTGF, IL4, MIP-1b were significantly elevated in NPDR patients when compared to PDR patients. Fragments of CTGF were identified more abundant in patients with PDR when compared to NPDR. Only three microRNAs were differentially expressed in the normal group when compared with either NPDR (decreased), namely miR-133a (p Disclosures No relevant conflicts of interest to declare.

Introduction Diabetic retinopathy is the most severe of several ocular complications of diabetes. Diabetes afflicts approximately 6% of the US population, thus retinopathy remains an important problem (PMID: 14702427). Our laboratory has provided evidence of the significant role of a pro-inflammatory axis during chronic inflammation (PMID: 19485899). The axis is comprised of TSP1-TGFβ-CTGF. We now provide evidence of an additional player within the axis, namely IL-4. A series of biochemical measurements were used to assess the role of the above mentioned axis in the progression of disease, from non-proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR). Methods Institutionally approved IRB and informed consent allowed this prospective study to recruit a total of 40 individuals from a vulnerable population disproportionately afflicted by this disease and representative of the population that we served in North Philadelphia with type-2 diabetes. Western-blotting technique was utilized to document the presence of fragments from CTGF in plasma, which have been shown in vitreous fluid to be angiogenic (PMID: 14988298). Multiplexed protein profiling on microarrays by rolling-circle amplification was employed to determine IL-4, IL-13, and MIP-1β levels. Commercially available ELISA was the method to determine the plasma concentration of TSP1, TGFβ and CTGF. Results and Discussion TSP1 (p Disclosures: No relevant conflicts of interest to declare.

We have previously demonstrated a low-affinity (0.8 microM, non-covalent complex formation between high-molecular-mass kininogen (HK) and plasminogen (Plg) which prevented Plg interaction with glioma and endothelial cells. We have now extended our previous observations by exploring the potential complex formation between Plg and low-molecular-mass kininogen (LK) and between LK and HK with Plg cleaved with human neutrophil elastase (HNE). Plg cleavage by HNE (PlgHNE) yielded kringles 1-3, kringle 4 and mini-plasminogen. PlgHNE was subjected to SDS/PAGE under non-reducing conditions, followed by western blotting, and incubated with either 125I-HK or 125I-LK. Autoradiograms revealed that 125I-HK bound to miniplasminogen and to kringles 1-3 but not to kringle 4 and the presence of 10 mM 6-aminohexanoic acid (Ahx) disrupted only the interaction with kringles 1-3. In contrast, 125I-LK bound to miniplasminogen but not to kringles 1-3 or 4 and Ahx had no effect at all. The complex formation of either HK (0.67 microM) or LK (3 microM) with Plg (1.5 microM) did not affect its conversion to plasmin by tissue plasminogen activator (t-PA) (10 U/ml) in the presence of a tissue plasminogen stimulator (0.14 microM). However, the rate of conversion of plasminogen to plasmin by t-PA was affected when platelets were added to the reaction mixture. Since HK (0.83 microM) has been shown to inhibit plasmin-induced platelet aggregation, we investigated whether this inhibitory property is found within the heavy chain shared by HK and LK. We found that LK inhibited plasmin-induced platelet aggregation, but a 4-fold molar excess was required when compared to HK. Compared to plasmin, 3-5-fold molar excess of miniplasmin is required to induce platelet aggregation, indicating the important role of kringles 1-3 for plasmin interactions with these cells. These results indicate that HK and LK-mediated inhibition of plasmin-induced platelet aggregation is likely due to complex formation with kringle 5 without interfering with plasmin's active site. We found an additional interaction between HK and kringles 1-3 enhancing the inhibitory effect, presumably by interfering with plasmin's interaction with platelets. This HK and LK-associated modulation of plasmin-induced platelet aggregation may serve as a template to develop synthetic peptides as novel therapeutic agents to prevent some of the plasmin-associated thrombocytopenia seen during thrombolytic therapy.

A specific plasma kallikrein inhibitor, Bz-Pro-Phe-boroArg (P8720), was used to define the relationship between the kallikrein-kinin (K-K) system and acute intestinal inflammation induced by bacterial peptidoglycan-polysaccharide (PG-APS) in Lewis rats. Group I received human serum albumin (HSA) intramurally in the intestine and was treated with HSA. Group II received PG-APS and was treated with P8720. Group III received PG-APS and was treated with HSA. P8720 attenuated the decrease of high-molecular-weight kininogen and factor XI activity (group II vs group III,P

Abstract 3268 Prasugrel is a very potent and effective P2Y12 receptor antagonist, used primarily to prevent thrombotic events in patients managed for percutaneous coronary intervention (PCI). It is not recommended as prophylaxis antithrombotic therapy because prasugrel increases patient's risk of bleeding. Even though indication of prasugrel treatment has been limited to the PCI procedure, there are not reports of the effects of prasugrel in patients with associated inflammatory diseases that undergo for PCI. Thus, we evaluate the effects of prasugrel, for 21 days, in a peptidoglycan polysaccharide (PG-PS)-induced arthritis rat model. Lewis rats were randomly separated into four groups from which only half of the animals were induced with PG-PS. There was a significant increase in the inflammatory changes observed in the PG-PS-induced animals treated with prasugrel compared to the PG-PS-induced control including a) increase joint diameter associated with visible signs of inflammation (during days 4 to 6 [p Disclosures: No relevant conflicts of interest to declare.

Abstract 3287 Clopidogrel is a widely used anti-thrombotic drug that prevents platelet activation by irreversibly antagonizing P2Y12 receptor. This drug could be co-administrated in patients afflicted with inflammatory conditions, such as rheumatoid arthritis and lupus. Hence it is of importance to determine if blocking P2Y12 receptors has any therapeutic benefit during the inflammatory response. Therefore, we evaluated the effect of clopidogrel using an animal model of chronic inflammation. Genetically susceptible Lewis rats were injected intraperitoneally with peptidoglycan polysaccharide (PGPS) to develop arthritis and followed for 21 days. Interestingly, we noticed an increased level of inflammation in induced arthritis animals treated with clopidogrel compared with induced arthritis animals alone, including a) increased in joint diameter between day 15 and 21 (p Disclosures: No relevant conflicts of interest to declare.

Abstract 3275 Background: Diabetic retinopathy (DR) is a progressive disease that affects over 4 million people in the United States and is one of the leading causes of blindness. The pathophysiology of the early events (no evidence of DR) leading to diabetic retinopathy is still not fully understood. Recent studies have proposed that levels of TSP1 (Arch Ophthalmol 127:507, 2009) and CTGF (Diabetes Care 27:758, 2004) may play a role in shifting the angiogenic balance and pathogenesis of DR. We postulate that during acute and chronic inflammation, as seen in type-2 diabetes a pro-inflammatory axis comprised by TSP1, transforming growth factor-beta (TGF-b) and CTGF may play a significant role in the progression of NPDR to PDR. Methods: This is a prospective control study comprised to date of: a) six (6) human subjects with NPDR, b) eleven (11) subjects with PDR, c) five (5) subjects without type-2 diabetes but in need of pars plana vitrectomy, and d) seventeen (17) normal human volunteers. Plasma samples were obtained from all groups (a-d) after signing an informed consent approved by our institution's IRB committee. Vitreous fluid (VF) was collected from subjects in b and c. Cytokine profile was determined by Multiplex Assay. TSP1, TGF-b and CTGF were assessed by commercially available ELISA assays and visualized by Western-blotting technique. Results: TNFa in conjunction with other pro-inflammatory cytokines was found elevated in NPDR subjects when compared to subjects in control groups (c-d, P Disclosures: No relevant conflicts of interest to declare.

Abstract 4721 The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists. The active metabolite of clopidogrel, a widely used anti-thrombotic drug, irreversibly antagonizes the P2Y12 receptor. However, treatment with clopidogrel has been associated with joint inflammation. In addition, Boilard E. et al identified micro-particles generated by activated platelets in joint fluid from rheumatoid arthritis (RA) patients. In this study we investigated whether platelet activation leads to changes in inflammatory responses using a rat model of erosive arthritis. Here, we evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of eight rats each: untreated group, animals receiving clopidogrel (clopidogrel group), animals receiving PG-PS (PG-PS group), and animals receiving PG-PS as well as clopidogrel (PG-PS + clopidogrel group). There were significant differences between the PG-PS + clopidogrel treated group when compared to PG-PS treated group including: increased joint diameter [4.24 ± 0.73 vs. 2.70 ± 1.09; p0.05], and elevated plasma levels of pro-inflammatory cytokines such as IL-1beta, IF gamma, IL-6. Plasma levels of anti-inflammatory cytokine IL-10 were significantly lower in PG-PS + clopidogrel treated group compared to PG-PS group alone. Histological observations indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation and decrease in bone mass upon treatment with clopidogrel in PG-PS-induced arthritis animals. Accordingly to the clinical, histological and cytokine results, we conclude that clopidogrel has a pro-inflammatory effect in PG-PS-induced arthritis animal model and this might implicate that P2Y12 receptor antagonism might enhance inflammatory responses. Disclosures: No relevant conflicts of interest to declare.

Abstract 4320 Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA are at an increased risk to develop cardiovascular diseases and the precise mechanism(s) of action remain obscure. This laboratory has proposed the existence of a pro-inflammatory axis in RA comprised by thrombospondin-1 (TSP1), transforming growth factor-beta (TGF-b) and CTGF. The present study evaluated plasma levels of TSP1, TGFb and CTGF in patients with RA by ELISA as well as specific cytokines and chemokines. CTGF plasma levels in RA patients (9.2 pg/ml mean, range 3.44–17.08) were found significantly increased (P Disclosures: No relevant conflicts of interest to declare.

Introduction: There is evidence in the literature indicating that activated factor X (FXa), thrombin and the complex of tissue factor (TF)-activated factor VII (VIIa) can induce upregulation of gene expression of connective tissue growth factor (CTGF) on human fibroblasts [JBC2000; 275]. However, there are no reports demonstrating the ability of human fibroblasts to assemble the prothrombinase complex on their cell-surface. Previously, we have demonstrated that a monocyte/neutrophil precursor cell line (HL60) was capable to assemble the prothrombinase complex on its cell surface, a reaction that was favored by the presence of thrombospondin-1 (TSP1) [Thromb. Res.2005; 115]. Objectives: We hypothesized that human fibroblasts due to their ability to express constitutively TF on their cell surface may be able to assemble the prothrombinase complex leading to cell signaling associated with upregulation of CTGF through the TGFβ pathway. Methods: Cultured human HS-68 fibroblasts, between four to ten passages, were incubated systematically with components of the prothrombinase complex, namely purified FVII (5nM), factor X (5nM), Factor V (45nM) prothrombin (1.4μM) and Ca++ (2mM). Generation of FXa and thrombin were measured with specific chromogenic substrates for FXa and thrombin. In addition, the reactions were assessed in the presence and absence of exogenous purified TSP1 (20nM). The signaling pathway potentially triggered by serine proteases generated (FXa and thrombin) were evaluated by the use of Western-blotting Analysis with specific antibodies directed to phosphorylated transcription factors. Results: Pre-incubation of FVII with cultured fibroblasts lead to its activation by cell-surface expressed TF, which in turn in the presence of FX, FV, FII (prothrombin) and Ca++ lead to the generation of FXa (9.7 ± 0.77 nM) and thrombin (7.99 ± 0.04 U/mL x10−3) as judged by efficient hydrolysis of specific chromogenic substrates. Addition of activated FV (FVa) but not FX after pre-incubation of FVII with cultured fibroblasts (under these experimental conditions) was an absolute requirement for the reactions to take place. Addition of TSP1 significantly enhanced thrombin generation (23.3 ± 0.69 U/mL x10−3 p In summary, TF expressed by normal human fibroblasts activates FVII and subsequently FX in the presence of FVa, prothrombin and Ca++. Generation of FXa and thrombin is associated with a downstream effect leading to upregulation of CTGF, independently of the TGFβ pathway. TSP1 not only promotes thrombin generation on the surface of human fibroblast but alone can induce CTGF up-regulation via the TGFβ pathway. This mechanism may play an important role in human diseases associated with fibrosis and with atherosclerosis.

Objective. To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods. Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-APS). Animals were divided into four groups:negative control and groups receivingno treatment,treatment with a TSP1-derived peptide andtreatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. Results. Histological data indicated that TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunohistochemical and RT-PCR analyses showed an increase of the CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to TSP1-derived peptide treated animals. By immunohistochemistry, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. Conclusion. Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues. Vasculature measurements at the perisynovial tissue of the TSP1-derived peptide treated (DT) and scramble peptide treated (DS) animals after day 16 PG-APS-induced erosive arthritis. Vascular Measurements Disease TSP1-derived peptide treated (DT) Disease scrambled peptide treated (DS) P Value Vascular area mean±SEM (μm2) 77.41 ± 7.96 μm2 336 ± 49.21 μm2 p=0.0008 Vessel wall thickness (μm) 3.53 ± 0.21μm 8.92 ± 0.48;μm p

Rheumatoid Arthritis (RA) is a chronic, autoimmune disease that affects a vast population worldwide with women being afflicted three times more than men. There is evidence for an increased risk of cardiovascular events in RA patients compared to the general population. These cardiovascular events may be associated with the chronic inflammatory state in which activation of coagulation leads to thrombin generation. Our laboratory has evidence that thrombospondin-1 (TSP1), an adhesive molecule that plays a major role in RA, promotes thrombin generation on the surface of a monocytic cell line (Isordia-Salas et al, Thromb Res.2005;116(6)). We also have documented that disrupting the TSP1 interaction on human neutrophils prevents the development of erosive arthritis in an experimental animal model (Manns et al, Arthritis and Rheumatism54(8), 2006). This observation is mediated by a novel pathway whereby TSP1 induces the up-regulation of Connective Tissue Growth Factor (CTGF). Therefore, to assess whether our in vitro and in vivo observations can be extrapolated to human disease, blood samples were collected from 20 patients afflicted with rheumatoid arthritis and 13 from healthy donors which served as the negative control. Plasma samples were separated and analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to determine the levels of transforming growth factor beta (TGF-β), protrombin F1+2 fragments (F1+2) and thrombospondin-1, and by multiplexed cytokine protein profiling on microarray by rolling-circle amplification (RCA) to determine cytokine levels. The F1+2 plasma levels showed an elevated trend in the RA group (p=0.06). TSP1 plasma levels were significantly increased in the RA group compared to the normal control (p=0.0004). Pro-inflammatory cytokine levels including IL-1β (p=0.0365), IL-6 (p=0.0029), TNF-α (p=0.0339), interferon-inducible protein 10 (p=0.0003) and macrophage inflammatory protein -1α (p=0.0012) were found elevated in the RA group compared to the normal control group. Some regulatory cytokines such as transforming growth factor-β (0.06), interferon-gamma (p=0.06) and IL-13 (p=0.22) showed no statistically significance between groups, but all of them showed a trend for higher circulation levels in plasma in the RA group. In summary, comparison between normal individuals and RA patients showed an increase in the levels of cytokines in the RA afflicted patients confirming what has been reported in the literature. We were able to correlate an increase in proteolytic factors and TSP1 levels in the RA patients with an increase of pro-inflammatory cytokines. Further studies are needed to elucidate why TSP1 acts as a pro-inflammatory molecule on the neutrophil surface of the RA patients.

There is evidence of shortening of the APTT in patients afflicted with and without cancer, and cancer is a well known underlying condition associated with hypercoagulability. Increased levels of coagulation factors such as VIII, IX, XI, II, and fibrinogen have been demonstrated as independent risk factors for venous thromboembolism (VTE). However, there is evidence that high levels of circulating factor VIII levels are not the only determinant for a shortened APTT (Blood 2004; 104:3631) and likely the same case for the other factors including fibrinogen levels. A shortened APTT in a patient with cancer with no evidence of a thrombotic event could be of clinical value in particular when the cost of measuring individual coagulation factors previously identified as risk factors for VTE would be very high. A lack of understanding of the underlying mechanism leading to a shortened APTT in the absence of VTE limit its clinical use, particularly for the clinical management of the patient presenting at admission with this phenomenon in association with cancer. In this case-control prospective study, we have investigated a potential mechanism associated with a shortened APTT in patients with cancer without evidence of thrombosis. The rationale for this study was a previous observation supporting a functional role of TSP1 in the generation of thrombin on a cell surface (Thrombosis Research 2005; 116: 533). The study was comprised of 69 human subjects subdivided in three separate groups, Group I, was constituted by 23 normal human volunteers, Group II included 23 patients without a shortened APTT and cancer, Group III was comprised of 23 patients with cancer with a shortened APTT (see table). Groups I and II were matched with group III by age and gender. In addition patients in Group II were matched with Group III by the type of cancer. Laboratory measurements included APTT, D-Dimer, soluble E, L, and P-Selectins as well as TSP1. Platelet and neutrophil counts were determined by automated methods. Laboratory measurements demonstrating a significant difference in Group III when compared with Group I and II were P-Selectin and TSP1. These results were independent of the platelet count in Group III. However the significant elevated circulating levels in plasma of P-Selectin in Group III are evidence supporting platelet activation. There was a trend for higher levels of D-Dimer in Group III (P < 0.17) when compared with Group II (P< 0.76), in accordance with previous studies reported in the literature. In summary, this prospective study demonstrates a potential association of a shortened APTT in patients with cancer with elevated circulating levels of soluble P-Selectin and TSP1. Our laboratory is currently investigating in more detail this interesting finding as well as the prospective clinical follow up of patients included in Groups II and III. | Parameter | Group I | Group II | Group III | P Value | |:------------------------------------ | -------- | -------- | --------- | ------- | | Normal Range APTT: 24.2–35.3 seconds | | aPTT | 29.3±2.7 | 28.2±2.3 | 23±1.0 | 0.01 | | sP-Selectin | 23.2±4 | 25.4±9 | 44.0±11 | 0.001 | | TSP1 | 382±39 | 871±496 | 1246±295 | 0.001 | Plasma Determinations

Gene expression of CTGF, a proangiogenic molecule, is up-regulated by serine proteases (FXa) and thrombin (JBC 275, 2000). We have demonstrated that thrombospondin-1 (TSP1) plays a major role in the assembly of the prothrombinase complex on the surface of neutrophils (PMNs) and that PMNs contain the message for CTGF. Rheumatoid arthritis is a chronic inflammatory disease associated with leukocyte adhesion and extravasation with angiogenesis as its hallmark. There is evidence indicating that TSP1 plays a major role in the pathophysiology of RA (JI 171, 2003). In this genetically susceptible rodent model of RA, a single intraperitoneal (i.p.) injection of peptidoglycan-polysaccharide induces an inflammatory immune response characterized by increased joint diameters in a biphasic manner. The acute phase occurs during the first five days with spontaneous reactivation around day 12 resulting in a progressive and irreversible chronic phase. We tested a well-characterized synthetic peptide derived from TSP1-type 3 repeats under three different therapeutic modalities in the experimental model of RA. First, peptide treatment was administered intravenously (i.v.) during the first five days of the experimental protocol representing the acute phase. Second, peptide treatment was administered i.v. during the acute phase but then i.p. every other day during the chronic phase of the experimental protocol. Third, peptide treatment was administered daily i.p. during the acute phase and every other day during the chronic phase. At all times a positive control group was included as well as a negative control group receiving a scrambled peptide. Total RNA was isolated from the ankle joints of the animals and RT-PCR was performed using specific primers for CTGF and G3PDH. Integrated density values were measured and the ratio of CTGF/G3PDH assessed. Immunohistochemical analysis of CTGF was also performed using Bioquant image analysis software to quantitate CTGF immuno expression. In peptide-treated animals CTGF expression in the cells of the synovial lining was significantly decreased when compared to the disease untreated group. Peptide untreated positive control group was associated with upregulation of the CTGF gene expression (2.53±0.33) in the ankle tissue and localized to the synovial membranes as judge by the immunochemical studies (8.7±0.77). In contrast, peptide treatment downregulated CTGF gene expression (0.9±0.41, p

1. One can accurately predict the contribution of each inhibitor to the total inactivation of an enzyme in plasma once its pseudo-first-order reaction rate constant and concentration are known. 2. Because the mechanism of augmentation of the inactivation rate of an enzyme by ATIII occurs via formation of an ATIII-heparin complex, the degree of potentiation can be predicted by knowing the binding capacity (sites per mole) of the heparin preparation and the concentration of heparin in the reaction (to calculate the concentration of the ATIII-heparin complex). 3. The augmentation by heparin of the inactivation rate of a particular enzyme by ATIII is dependent upon the presence of other enzymes with higher kassoc, since these would strongly compete for the ATIII-heparin complex. 4. In a plasma environment, using therapeutic levels of heparin, there is no augmentation of the inactivation rate of any of the contact enzymes.