Thrombin Infusion Research Articles

Continuous thrombin infusion leads to a bleeding phenotype in sheep

BackgroundIn addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo. MethodsThrombin (0.0004-0.42IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n=5 animals; 15 experiments). In the dose verification study animals received 0.42IU/kg/min of thrombin vs. saline solution in a cross-over design (n=3 animals; 7 experiments). ResultsThrombin at an infusion rate of 0.42IU/kg/min decreased fibrinogen levels by 75% (p<0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p=0.006), prolonged thrombin time by 70% (p=0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p<0.05) and decreased the activity of the coagulation factors VIII and XIII (p<0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p=0.019) and induced a 65% decrease in maximal clot firmness (p<0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism. ConclusionProtracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species.

Decreased lung hyaluronan in a model of ARDS in the rat: Effect of an inhibitor of leukocyte elastase

Background. Hyaluronan (HA) is a component of the extracellular matrix in lung tissue and is normally present at low concentrations in blood. HA is rapidly cleared from blood by the liver. Increased concentrations of plasma HA have been found in patients with acute respiratory distress syndrome (ARDS). We investigated changes in HA levels in plasma, bronchoalveolar lavage fluid (BALF), and lung, and their relationship to pretreatment with a leukocyte elastase inhibitor in a rat model of ARDS.Methods. Rats were randomly assigned to three groups: control, thrombin, and thrombin plus elastase inhibitor. By use of a radiometric assay, HA was measured in lungs, BALF, and plasma. Tissue samples from the lungs were stained for HA and examined microscopically. Liver circulation and cardiac output were monitored using radiolabeled microspheres.Results. Infusion of thrombin produced a pronounced increase in wet weight to dry weight ratio, and relative lung water content. This increase was blunted by a leukocyte elastase inhibitor. A decrease in lung HA and increases in both BALF and plasma HA were found. The leukocyte elastase inhibitor counteracted not only the decrease in lung tissue HA, but also the increase in plasma HA. Histologically, there was decreased HA-staining of peribronchial and perivascular areas in the injured rat lung. Decreased liver perfusion was observed after infusion of thrombin.Conclusions. The decrease in lung HA may be involved in the development of pulmonary edema in this ARDS model, and leukocyte elastase may be one cause of this decrease. In addition, an elevated plasma HA level may be an indicator of lung injury.

Deficiency of Superoxide Dismutase Impairs Generation of Activated Protein C and Enhances Susceptibility to Experimental Thrombosis in Mice

Abstract 535In vitro studies have suggested that reactive oxygen species such as superoxide can produce several potentially prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin that impairs its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo. Using a murine model that is genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), we tested the hypothesis that lack of superoxide dismutase enhances susceptibility to thrombosis. Additionally, we investigated the mechanisms of superoxide-enhanced thrombosis. First, we examined the susceptibility to carotid artery thrombosis in a photochemical injury model. We found that Sod1−/− mice formed stable occlusions significantly faster than Sod1+/+ or Sod1+/− mice (P<0.05). Further, using an inferior vena cava (IVC) stasis method we observed that Sod1−/− mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 compared with Sod1+/+ or Sod1+/− mice). These findings suggest that deficiency of SOD1 leads to increased susceptibility to both arterial and venous thrombosis in mice. To address the mechanism of accelerated thrombosis, we first examined activation of platelets in response to multiple agonists using flow cytometry. After activation by thrombin (0.5 U/ml) and convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen to activated platelets were observed between Sod1−/−, Sod1+/+, or Sod1+/− mice, suggesting that increased susceptibility to thrombosis in Sod1−/− mice is not platelet mediated. Next, we measured expression of TF mRNA in lung by real time qPCR. TF mRNA levels in Sod1−/− mice were similar to those in Sod1+/+ mice, suggesting that deficiency of SOD1 does not influence TF expression in mice. Finally, we measured the activation of protein C in vivo in response to infusion of thrombin (40 U/Kg). Generation of activated protein C was significantly lower in Sod1−/− mice compared with Sod1+/+ mice (P<0.05). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1−/− mice compared with Sod1+/+ mice (P=0.4 and 0.6 respectively), suggesting that altered generation of activated protein C in Sod1−/− mice may be related to a direct oxidative effect on thrombomodulin rather than to decreased expression of thrombomodulin or EPCR. We conclude that lack of SOD1 in mice accelerates thrombosis and impairs the protein C anticoagulant response to thrombin. Disclosures:No relevant conflicts of interest to declare.

Human Thrombomodulin Knock-In Mice Reveal Differential Effects of Human Thrombomodulin on Thrombosis and Atherosclerosis

We sought to develop a murine model to examine the antithrombotic and antiinflammatory functions of human thrombomodulin in vivo. Knock-in mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knock-in mice exhibited decreased protein C activation in the aorta (P<0.01) and lung (P<0.001). Activation of endogenous protein C following infusion of thrombin was decreased by 90% in knock-in mice compared with wild-type mice (P<0.05). Carotid artery thrombosis induced by photochemical injury occurred more rapidly in knock-in mice (12±3 minutes) than in wild-type mice (31±6 minutes; P<0.05). No differences in serum cytokine levels were detected between knock-in and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E-deficient mice and fed a Western diet, knock-in mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis. Expression of human thrombomodulin in place of murine thrombomodulin produces viable mice with a prothrombotic phenotype but unaltered responses to systemic inflammatory or atherogenic stimuli. This humanized animal model will be useful for investigating the function of human thrombomodulin under pathophysiological conditions in vivo.

Proteinase-Activated Receptors 1 and 2 Exert Opposite Effects on Renal Renin Release

Proteinase-activated receptors (PARs) 1 to 4 are highly expressed in the kidney and are involved in the regulation of renal hemodynamics and tubular function. Since intravascular infusion of the proteinase thrombin, which activates PARs, has been shown to decrease plasma renin activity in rats, we investigated the effects of the respective PAR subtypes on renin release using the isolated perfused mouse kidney model. Thrombin dose-dependently reduced perfusate flow and inhibited renin secretion rates (RSRs) that had been prestimulated by the β-adrenoreceptor agonist isoproterenol. The suppression of RSRs was prevented by the selective PAR1 inhibitor SCH79797, and direct activation of PAR1 by TFLLR mimicked the effects of thrombin on RSRs and vascular tone. Moreover, TFLLR suppressed the stimulations of RSRs in response to the loop diuretic bumetanide, to prostaglandin E(2), or to a decrease in renal perfusion pressure but not in response to a reduction in extracellular calcium. The PAR2-activating peptide SLIGRL concentration dependently increased RSR and perfusate flow. The stimulation of RSRs by SLIGRL was markedly attenuated by N(G)-nitro-L-arginine methyl ester, suggesting an NO-dependent mechanism. Activation of PAR4 by AYPGKF did not modulate RSRs or perfusate flow. PAR1 and PAR2 immunoreactivity were detected in the juxtaglomerular region and were colocalized with renin immunoreactivity. Our data provide evidence that PAR1 activation inhibits renal renin secretion and induces renal vasoconstriction, whereas PAR2 activation stimulates renin release and induces vasodilation mainly via the release of NO.

Thrombose coronaire distale compliquant une leucémie aiguë lymphoblastique traitée par asparaginase

A 53-year-old man is treated by l-asparaginase for an acute lymphoblastic leukaemia. He received anti thrombin infusions. A systematic electrocardiogram showed an asymptomatic subepicardium ischemia without troponin elevation. Echocardiography and heart magnetic resonance imaging showed an apical thrombus facing a zone of myocardial necrosis. A thrombus regression was observed under anticoagulation. Atypical and asymptomatic coronary thrombosis may occur following l-asparaginase treatment. Regular electrocardiogram monitoring is proposed along this treatment. Arterial thrombosis associated with anti tumor chemotherapies are reviewed.

Thrombin as a multi-functional enzyme

Thrombin is the central protease in the coagulation cascade and one of the most extensively studied of all enzymes. In addition to its recognised role in the coagulation cascade and haemostasis, thrombin is known to have multiple pleiotropic effects, which mostly have been shown only in in vitro studies: it plays a role in inflammation and cellular proliferation and displays a mitogen activity on smooth muscle cells and endothelial cells, predominantly by activation of angiogenesis. In vivo , thrombin effects were examined in animal models of intravenous or intraarterial thrombin infusion. An extensive literature search regarding in vivo data showed that i) thrombin administered as a bolus causes microembolism, ii) thrombin infused slowly at steady-state conditions (up to 1.6 U/kg/min) leads to bleeds but not to intravascular clotting, iii) large quantity of thrombin infused at low rates (0.05 U/kg/min) does not have any measurable effect, and iv) thrombin increases vascular permeability leading to tissue damage. Although several decades of research on thrombin functions have provided a framework for understanding the biology of thrombin, animal and human studies with use of newer laboratory techniques are still needed to confirm the pleiotropic thrombin functions shown in in vitro studies.

Noninvasive Treatment of Deep Venous Thrombosis Using Pulsed Ultrasound Cavitation Therapy (Histotripsy) in a Porcine Model

Purpose This study evaluated histotripsy as a noninvasive, image-guided method of thrombolysis in a porcine model of deep vein thrombosis. Histotripsy therapy uses short, high-intensity, focused ultrasound pulses to cause mechanical breakdown of targeted soft tissue by acoustic cavitation, which is guided by real-time ultrasound imaging. This is an in vivo feasibility study of histotripsy thrombolysis. Methods and Materials Acute thrombi were formed in the femoral vein of juvenile pigs weighing 30–40 kg by balloon occlusion with two catheters and thrombin infusion. A 10-cm-diameter 1-MHz focused transducer was used for therapy. An 8-MHz ultrasound imager was used to align the clot with the therapy focus. Therapy consisted of five cycle pulses delivered at a rate of 1 kHz and peak negative pressure between 14 and 19 MPa. The focus was scanned along the long axis of the vessel to treat the entire visible clot during ultrasound exposure. The targeted region identified by a hyperechoic cavitation bubble cloud was visualized via ultrasound during treatment. Results Thrombus breakdown was apparent as a decrease in echogenicity within the vessel in 10 of 12 cases and in 7 cases improved flow through the vein as measured by color Doppler. Vessel histology found denudation of vascular endothelium and small pockets of hemorrhage in the vessel adventitia and underlying muscle and fatty tissue, but perforation of the vessel wall was never observed. Conclusions The results indicate histotripsy has potential for development as a noninvasive treatment for deep vein thrombosis.

An experimental model to study isolated effects of thrombin in vivo

Background In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We hypothesized that protracted intravenous infusion of thrombin at steady state will allow to study isolated thrombin effects in vivo. Methods Thrombin (0.05-0.9 U/kg/min) was continuously infused in Sprague Dawley rats over five hours (n = 38). The study consisted of three parts: dose escalation (n = 21), dose verification (n = 5) and a parallel group study to investigate whether thrombin effects can be antagonised by concomitant infusion of lepirudin (n = 12). Results A thrombin dose of 0.9 U/kg/min decreased platelet counts by 70% compared to the control group (median 230 × 10^9/L vs. 752 × 10^9/L; p = 0.041). In accordance, infusion of 0.9 U/kg/min of thrombin decreased fibrinogen level by 75% compared to the control group (56 mg/dl vs. 220 mg/dl; p = 0.046). Cumulative thrombin doses of ≥ 0.1 U/kg/min caused bleedings but not thromboembolic events. Thrombin at doses ≥ 0.15 U/kg/min was lethal in four cases (30%). Platelet counts and fibrinogen levels after thrombin infusion correlated with bleeding events and mortality. Administration of thrombin at cumulative doses of 0.3-0.9 U/kg/min was associated with a 3 to 6.5 -fold increase in IL-6 levels (139-306 pg/ml vs. 47 pg/ml, p < 0.05). In contrast, thrombin infusion did not alter other markers of inflammation (IL-10, MCP-1 or TNF-alpha). In addition, lepirudin prevented thrombin- induced thrombocytopenia. Conclusion Protracted intravenous infusion of thrombin offers a new experimental model, where consumption of fibrinogen and platelets correlates with bleedings and mortality. Infusion of thrombin increased only IL-6 levels but not other cytokines.

Thrombin Mediates Severe Neurovascular Injury During Ischemia

Cerebral ischemia initiates cascades of pathological events such as edema, blood-brain barrier breakdown, and tissue degeneration. Thrombin activation is a key step in coagulation, and thrombin has recently been shown to mediate endothelial permeability and cellular toxicity in vitro. We examined the effect of thrombin on vasculature during ischemia in vivo. Focal ischemia was induced in adult Sprague-Dawley rats by occlusion of the middle cerebral artery for 4 hours followed by a short period of reperfusion. High-molecular-weight fluorescein isothiocyanate-dextran was injected before surgery to label the severe vascular disruption. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to identify dying cells, which were quantified with manual counts. Intra-arterial thrombin or intravenous thrombin inhibitors were infused during ischemia and reperfusion. Infusion of thrombin (3 U/kg) intra-arterially during ischemia greatly enlarged the volume of severe vascular disruption, as visualized by fluorescein isothiocyanate-dextran extravasation (P<0.05). Thrombin also promoted blood-brain barrier leakage of IgG during ischemia. Vascular disruption was blocked by intravenous infusion of the direct thrombin inhibitor argatroban (1.69 mg/kg, P<0.05). Greater numbers of dying cells were found in regions of severe vascular disruption, and interventions that reduced vascular leakage also reduced the numbers of dying cells. Thrombin mediates severe vascular disruption during ischemia and thrombin inhibitors may partially ameliorate vascular disruption. Further work is needed to establish whether thrombin, entering parenchyma due to increased vascular permeability, augments neurotoxicity during ischemia.

A novel system for the investigation of microvascular dysfunction including vascular permeability and flow-mediated dilatation in pressurised human arteries

Introduction Adverse drug reactions may be manifested through changes in microvascular function (e.g. angioedema) or by subtle modification of the mechanisms controlling vascular tone, such as flow-mediated dilatation. Until now the early detection of such adverse drug reactions has been hampered by the lack of a predictive in vitro model. This in vitro model can be utilised to test potential effect of drugs on the normal responses of the vascular system. Methods The PM-1, a new automated perfusion myograph, allows detection of the external and internal dimensions of tubular biological structures and regulates both the intraluminal pressure and flow independently. Drugs can be infused intraluminally or extraluminally (by adding to the bathing solution) to determine effects on constriction, relaxation or modulation of vascular tone. The novel imaging system also facilitates the measurement of vascular permeability using dyes introduced intraluminally into the vessel. Results To assess effects on flow-mediated dilatation we increased flow rate in pressurised human subcutaneous arteries (< 500 μm diameter) in the absence and presence of various drugs. Increasing flow from 0.04 ml/min to 0.3 ml/min resulted in a 39 ± 3% relaxation of a U46619 pre-constriction (10 − 6 M). This was enhanced in the presence of Ivermectin and inhibited in the presence of 100 µM L-NAME (316 ± 169% and 16 ± 1% respectively).To assess effects on vascular permeability we infused albumin-bound Evans blue dye through the lumen of human subcutaneous arteries as a marker, in the absence and presence of a modulatory drug. Infusion of thrombin (0.5 units/ml) through the vessel lumen caused an 11.8% increase in vessel permeability compared to vehicle infusion. Conclusion The development of the PM-1 allows new drugs to be tested in relevant human or animal tissues at an early stage allowing crucial go/no-go decisions to be made early in development and giving a more complete picture of the overall effects of test compounds on vascular function.

Structural and Functional Studies to Define the Molecular Basis by Which Platelet Factor 4 (PF4) Increases Survival of Mice in Lipopolysaccharide (LPS)-Induced Endotoxicity

Our previous studies have demonstrated that platelet-released PF4 (chemokine CXCL4) promotes survival in a murine LPS-induced endotoxicity model, although the molecular basis for PF4's protective effects was not fully defined. We hypothesized that enhanced generation of cytoprotective activated protein C (APC) by PF4 might contribute to the molecular mechanism of PF4's beneficial effects in vivo, based on the observation that PF4 stimulates protein C (PC) activation by the thrombin/thrombomodulin complex both in vitro and in vivo. Here we show that PF4 in vitro affects human (h) PC activation in the presence of human thrombomodulin (hTM) in a bell-shaped concentration curve, i.e. stimulation at low, but inhibition at high PF4 concentrations with a peak around 3 μM. This curve is similar to that seen with PF4 for surface-bound heparin-induced thrombocytopenia (HIT) antigenicity, suggesting that similar complexes of PF4 with glycosaminoglycans (GAGs) that occur in HIT (termed ultralarge complexes (ULC)) are relevant to PF4's interaction with the hPC/hTM. Addition of heparin blocks PF4 increase of APC generation in a similar fashion as it does for surface-bound ULC. A PF4 variant PF4K50E that poorly forms PF4 tetramers requires 8-fold higher concentrations to enhance APC generation, supporting that PF4 tetramers are central for APC generation as they are for formation of ULC. Neither PF4 nor PF4K50E accelerated in vitro generation of APC in the presence of hTM that was depleted of its chondroitin sulfate chain, suggesting that PF4 binds to this domain on hTM. In vivo studies involving simultaneous infusions of PF4 and thrombin into PF4 knock out (mPF4−/−) mice showed that PF4 leads to enhanced mouse (m) APC generation not seen with infused PF4K50E, consistent with our in vitro studies. We then asked if surface heparan sulfate on the endothelial lining was necessary for the observed PF4 effect on in vivo mAPC formation. We studied mice with a Tie2-Cre conditional knock out of N-deacetylase-N-sulfotransferase-1 activity (NDST-1−/−) that have only 15% of normal endothelial cell surface heparan sulfate content using a similar thrombin/PF4 infusion model. We found that mAPC generation was accelerated by PF4 to the same extent both in NDST1−/−/mPF4−/− and mPF4−/− mice, suggesting that surface GAGs are not involved in the PF4 effect. We have also tested the in vivo effect of PF4 on mAPC formation in TM mutant (TMpro/pro) mice that have impaired capacity for APC formation to further demonstrate that PF4's positive effect in LPS endotoxic shock survival involves enhanced mAPC generation. Upon injection of high doses of thrombin (40 U/kg), mAPC levels are increased to the same extent in WT and TMpro/pro mice. After injection of low amounts of thrombin (8 U/kg), generation of mAPC was impaired in TMpro/pro as compared to WT mice. Concurrent infusion of PF4 increased mAPC formation in TMpro/pro mice after injection of low doses of thrombin, approximately equal to that seen in WT mice with no PF4 injected. As previously described, TMpro/pro mice had increased mortality after injection of LPS as compared to WT mice; however, with concurrent platelet PF4 overexpression, mortality decreased to that seen in WT mice, suggesting that the biological value of PF4 in LPS endotoxicity is related to its effect on the generation of APC. Thus, these studies support enhanced APC generation as the basis for the positive effect of PF4 on LPS endotoxicity and further define the molecular basis for increased APC generation by PF4 by forming ULC with the chondroitin sulfate domain of TM, but not with heparan sulfate on the vascular surface.

Response to Letter by Culp and Culp

Response: We greatly appreciate the thoughtful comments by Drs Culp and Culp. First, we would like to acknowledge the excellent work the authors have done describing the angiographic cerebral vascular anatomy of the rabbit.1 The first draft of our paper was submitted in November 2006 with several revisions before final acceptance.2 On resubmission, we failed to cite the article by Culp et al, which had appeared in the interim. Concern is raised about the extent of MCA occlusion in our model and that infusion of thrombin may lead to a greater degree of vascular occlusion compared to that seen in the typical human stroke where there is only segmental occlusion …

Endogenous platelet factor 4 stimulates activated protein C generation in vivo and improves survival after thrombin or lipopolysaccharide challenge

AbstractPharmacologic infusion of activated protein C (APC) improves survival in severe sepsis, and platelet factor 4 (PF4) accelerates APC generation in a primate thrombin-infusion model. We now tested whether endogenous platelet PF4 content affects APC generation. Mice completely deficient in PF4 (mPF4−/−) had impaired APC generation and survival after thrombin infusion, similar to the impairment seen in heterozygote protein C–deficient (PC+/−) mice. Transgenic mice overexpressing human PF4 (hPF4+) had increased plasma APC generation. Overexpression of platelet PF4 compensated for the defect seen in PC+/− mice. In both a thrombin and a lipopolysaccharide (LPS) survival model, hPF4+ and PC+/−/hPF4+ mice had improved survival. Further, infusion of hPF4+ platelets improved survival of wild-type mice after an LPS challenge. These studies suggest that endogenous PF4 release may have biologic consequences for APC generation and survival in clinical sepsis. Infusions of PF4-rich platelets may be an effective strategy to improve outcome in this setting.

Effects of membrane and soluble EPCR on the hemostatic balance and endotoxemia in mice

AbstractRecent studies have shown that endothelial protein C receptor (EPCR) polymorphisms and soluble EPCR levels are associated with thrombotic diseases. It is unknown whether membrane EPCR (mEPCR) heterozygosity and/or physiologically elevated sEPCR levels directly impact the hemostatic balance and the outcome of endotoxemia. In these studies, thrombin infusion experiments revealed that EPCR heterozygosity (Procr+/−) impaired protein C activation by approximately 30%. Infusion of factor Xa with phospholipid demonstrated that the Procr+/−genotype increased the coagulant response relative to wild-type mice. Challenge of the Procr+/− mice with lipopolysaccharide (LPS) did not significantly exaggerate their response compared with wild-type mice. We also generated mice in which one allele of full-length EPCR was replaced by sEPCR (Procrs/+). Compared with Procr+/− mice, Procrs/+ mice had 5-fold higher sEPCR and similar mEPCR levels. Procr+/− and Procrs/+ mice generated similar levels of activated protein C (APC) upon thrombin infusion. They also exhibited a similar coagulant response upon factor Xa/phospholipid infusion. Only supraphysiologic levels of sEPCR could influence protein C activation and exaggerate the coagulant response. In conclusion, mEPCR, but not physiologically elevated sEPCR, regulated protein C activation. Procr heterozygosity results in a mild increase of thrombosis tendency and little influence on the response to endotoxin.

Release of High Levels of Platelet Factor 4 (PF4) from Platelets Improves Survival after a Lethal Lipopolysaccharide (LPS) Challenge.

Infusion of activated protein C (APC) improves survival in sepsis. PF4 is a CXC chemokine predominantly expressed during megakaryopoiesis and stored in platelet alpha-granules whose biological function(s) are not well understood. We have shown that recombinant PF4 enhances APC generation by thrombin/thrombomodulin complexes both in vitro and in vivo. Would endogenous PF4 released from platelets activated during an inflammatory state similarly affect APC production? We addressed this issue using mice that were either completely devoid of PF4 (mPF4−/−) or had a 6-fold excess of human PF4 (hPF4+). Using a lethal LPS challenge model (O111:B4, 40 mg/kg IP), we examined whether platelets become activated and release PF4 during endotoxemia. Two hours after LPS injection, the platelet count in mice decreased to ~70% of baseline levels (p=0.006). Serum PF4, as measured by ELISA, also dropped to ~60% of baseline from 47±5 kU/ml to 30±5 kU/ml (p=0.002). At the same time, plasma PF4 level was increased by 20%, consistent with LPS resulting in PF4 release. The smaller increase than expected suggests that much of the released PF4 binds immediately to the surface of vascular cells. Consistent with this, we have observed higher accumulation of PF4 in mouse lungs after LPS injection compared to uninjected mice (990±220 and 660±120 U/mg, respectively, p=0.017). APC generation was assessed 10 min after thrombin infusion (80 U/kg. IV) as a measure of endogenous platelet PF4's effect in an inflammatory/procoagulant state. In mPF4−/− mice APC levels were 72% of that in wild type (WT) mice (p=0.0006) while in hPF4+ mice APC formation increased to 178% (p=0.003). Survival of mice 24 hrs after LPS (25 mg/kg) challenge was then examined. hPF4+ mice had a mortality rate of 9% compared to ~40% in both WT and mPF4−/− (p< 0.001). To examine the role of APC in this improved survival, we performed similar experiments with mice heterozygous for protein C deficiency (PC+/−). More PC+/− mice died 16 hrs after injection of 40 mg/kg LPS than WT mice (61% vs. 29% mortality respectively, p=0.005), while mortality for hPF4+/PC+/− mice was significantly lower (14%, p< 0.001 compared to PC+/− mice), supporting the hypothesis that the protective effect of PF4 is at least in part due to increased APC generation. Next we asked if infusion of platelets with high PF4 is protective in the LPS model. We injected either vehicle buffer or mPF4−/− or hPF4+ platelets (3×108 per 20 g mouse) into WT mice prior to treatment with LPS. Mortality of mice at 24 hrs after LPS injection with mPF4−/−platelet infusion was not significantly different than mice with buffer infusion (86% vs. 96% respectively, p=0.4), but mortality was significantly lower when hPF4+ platelets were infused (58% vs. 96%, p=0.01). Our results suggest that PF4 is released from platelets after an inflammatory stimulus and that this may have a positive physiological role by enhancing APC generation. High endogenous platelet PF4 levels may have a survival advantage after exposure to endotoxins. Infusion of platelets containing high levels of PF4 in sepsis may be a novel therapeutic strategy that warrants further investigation.

Thrombin effectuates therapeutic arteriogenesis in the rabbit hindlimb ischemia model: A quantitative analysis by computerized in vivo imaging

We report on an experimental mammalian controlled study that documents arteriogenic capacity of thrombin and utilizes computerized algorithms to quantify the newly formed vessels. Hindlimb ischemia was surgically invoked in 10 New Zealand white rabbits. After quiescence of endogenous angiogenesis heterologous bovine thrombin was intramuscularly injected (1500 units) in one hindlimb per rabbit (Group T). Contralateral limbs were infused with normal saline (Group C). Digital subtraction angiography (DSA) of both limbs was performed after thrombin infusion by selective cannulation of the abdominal aorta and digital images were post-processed with computerized algorithms in order to enhance newly formed vessels. Total vessel area and total vessel length were quantified. In vivo functional evaluation included measurements of blood flow volume at the level of the external iliac artery by Doppler ultrasonography both at baseline and at 20 days after thrombin infusion. Total vessel area and length (in pixels) were 14,713±1023 and 5466±1327 in group T versus 12,015±2557 and 4598±1269 in group C ( p = 0.0062 and 0.1526, respectively). Blood flow volumes (ml/min) at baseline and at 20 days after thrombin infusion were 25.87±11.09 and 38.06±11.72 in group T versus 26.57±11.19 and 20.35±7.20 in group C ( p = 0.8898 and 0.0007, respectively). Intramuscular thrombin effectuates an arteriogenic response in the rabbit hindlimb ischemia model. Computerized algorithms may enable accurate quantification of the neovascularization outcome.

Tumor Necrosis Factor-α Increases in the Brain after Intracerebral Hemorrhage and Thrombin Stimulation

The goals of this study were 1) to determine the effects of intracerebral hemorrhage (ICH) on brain tumor necrosis factor (TNF)-alpha levels, which are still controversial; 2) to investigate the role of TNF-alpha in ICH-induced brain injury; 3) to examine the effects of thrombin on brain TNF-alpha levels; and 4) to elucidate the role of TNF-alpha in thrombin-induced neuroprotection. Autologous whole blood and thrombin were injected into the right caudate of rats or mice. Brain TNF-alpha was then determined by enzyme-linked immunosorbent assay and immunohistochemistry. Brain edema and neurological deficits were also examined. Perihematomal TNF-alpha levels increased after ICH. ICH-induced brain edema was less in TNF-alpha knockout mice compared with wild-type mice (P < 0.05). Intracerebral infusion of thrombin also caused an increase in brain TNF-alpha levels. Thrombin preconditioning reduced thrombin-induced brain edema, but this effect was not blocked by a neutralizing TNF-alpha antibody. Increase of perihematomal TNF-alpha levels contributes to brain edema formation after ICH. Thrombin may be a major mediator of ICH-induced TNF-alpha production, but thrombin-induced brain tolerance may not be TNF-alpha mediated.

Endogenous Platelet PF4 Promotes In Vivo Activated Protein C (APC) Generation and Survival after Lethal Lipopolysaccharide Challenge in Mice: A Potential Physiologic Role for PF4.

Pharmacologic infusion of activated protein C (APC) improves survival in human sepsis and genetically induced deficiencies in APC generation increase mortality in a murine lipopolysaccharide (LPS) model of inflammation/sepsis. We have shown that the platelet α-granule-specific chemokine, platelet factor 4 (PF4), accelerates thrombin·thrombomodulin complex APC generation in vitro and in vivo when recombinant PF4 is pharmacologically co-infused in a primate thrombin infusion model system. Hypothesizing a potential physiologic role for PF4 in stimulating APC formation in vivo, we tested whether variations in the level of endogenous platelet PF4 content affect APC generation and also influence survival in the murine LPS challenge model. To stimulate APC formation and platelet PF4 release in mice, we injected murine thrombin (80U/kg IV). Ten min after thrombin injection into wildtype (WT) C57BL/6J mice, plasma APC levels, assessed by enzyme capture amidolytic assay, increased ~10-fold over basal levels. Injection of concurrent murine PF4 (7.5 mg/kg IV) further increased APC levels 2-2.6-fold. We then studied APC generation in mice that were either completely deficient in PF4 (PF4−/−) or transgenic mice that had 6-fold normal levels of human PF4 (hPF4+), both of which had been crossbred onto the same C57BL/6J background >10 times. Plasma APC generation by thrombin was 74 ± 6% in the PF4−/− mice (n = 7) compared to WT littermates (n = 15, p<0.1) and increased to 178 ± 49% in hPF4+ mice (n= 22, p< 0.003 compared to WT (n= 36)). Heterozygous protein C deficient (PC+/−) mice had 65 ± 21% of WT APC generation (n= 5, p= 0.05 compared to WT (n=7)), but APC generation was restored to 96 ± 6% level in hPF4+/PC+/− mice (n= 6, p= 0.88 compared to WT (n=12)). Only PF4−/− and PC+/− mice died during this thrombin injection trial with mortality rates of 30% and 58%, respectively. A lethal LPS challenge model (25 mg/kg IP) was used to test the susceptibility of mouse strains with varying PF4 levels. Significant numbers of WT and PF4−/− mice died before the end of the first day [(18 of 44 (40% mortality) and 6 of 21 (28% mortality), respectively], while none of the 19 hPF4+ animals died (p< 0.001). However, after the first day, mortality did not differ significantly between the groups and final mortality for the three groups was ~70%. In summary, these studies of mice with varying platelet PF4 suggest that endogenous PF4 plays a physiologic role to stimulate APC production. Moreover, mice with high platelet PF4 content appear to be protected from both thrombin- and LPS-provoked deaths. This suggests that PF4 plays a physiologic role to enhance APC generation, that patients with high PF4 levels may be intrinsically protected from sepsis and supports the possibility that PF4 infusions in patients with sepsis may be a viable therapeutic strategy.

Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin.

Previous studies have shown that blocking endothelial protein C receptor (EPCR)-protein C interaction results in about an 88% decrease in circulating activated protein C (APC) levels generated in response to thrombin infusion and exacerbates the response to Escherichia coli. To determine whether higher levels of EPCR expression on endothelial cells might further enhance the activation of protein C and protect the host during septicemia, we generated a transgenic mouse (Tie2-EPCR) line which placed the expression of EPCR under the control of the Tie2 promoter. The mice express abundant EPCR on endothelial cells not only on large vessels, but also on capillaries where EPCR is generally low. Tie2-EPCR mice show higher levels of circulating APC after thrombin infusion. Upon infusion with factor Xa and phospholipids, Tie2-EPCR mice generate more APC, less thrombin and are protected from fibrin/ogen deposition compared with wild type controls. The Tie2-EPCR animals also generate more APC upon lipopolysaccharide (LPS) challenge and have a survival advantage. These results reveal that overexpression of EPCR can protect animals against thrombotic or septic challenge.