Increased Clot Formation Research Articles

Altered clot formation and fibrinolysis in heart transplant recipients

Abstract Background Long-term survival after heart transplantation (HTx) is limited by cardiac allograft vasculopathy (CAV), a frequent accelerated form of coronary artery disease. The pathogenesis remains incompletely understood, but thrombosis may be involved. We aimed to investigate clot formation and fibrinolysis in HTx patients and relate the findings to prognosis. Methods This prospective cohort study included 69 patients (median 8.4 years after HTx) with angiographically documented CAV 0 (not significant, n=36), CAV 1 (mild, n=19), and CAV 2-3 (moderate to severe, n=14). Healthy individuals (n=69) served as age- and gender-matched controls. Using a plasma-based fibrin clot formation and lysis assay, we assessed the following clot properties off-aspirin: 1) clot formation reflected by peak absorbance, 2) fibrinolysis measured by clot lysis time, and 3) a combined measure of clot formation and lysis reflected by area under the curve (AUC). Major adverse cardiac events (MACE) included heart failure hospitalization, cardiovascular death, and significant CAV progression. Results Median follow-up time was 5.1 (2.1-5.9) years. Five CAV 2-3 patients were excluded as it was not considered safe to interrupt aspirin therapy. Five patients had missing values due to either lysis resistance or severely decreased fibrin formation, and four of these underwent MACE with CAV progression and/or heart failure during follow up. Peak absorbance was significantly higher in HTx patients compared with healthy controls (0.72 vs. 0.50 aggregation units (AU), p<0.0001). Further, a stepwise increment above reference range through CAV groups was seen; CAV 2-3 patients (0.85 ± 0.12 AU) versus CAV 1 patients (0.72 ± 0.17 AU, p<0.05) and CAV 0 patients (0.68 ± 0.27 AU, p=0.06). Clot lysis time was significantly longer in HTx patients compared with healthy controls (1275 vs. 733 seconds, p<0.0001). AUC was significantly increased compared with healthy controls (1399 vs. 547 AU x seconds, p<0.0001). Overall, clot formation and fibrinolysis was not related to MACE. Conclusion HTx-patients have increased clot formation and prolonged fibrinolysis compared with healthy controls. Further, HTx-patients have an increased combined measure of these parameters compared with healthy controls, suggesting imbalance between fibrin clot formation and fibrinolysis. None of the measured parameters were related to prognosis.Clot formation and fibrinolysis

Practice Algorithm of Rotational Thromboelastometry-Guided (ROTEM-Guided) Bleeding Management in Liver Transplantation.

Liver transplantation (LT) is frequently complicated by coagulopathy associated with end-stage liver disease, which is often multifactorial and associated with hemostatic disturbances affecting both the procoagulant and anticoagulant systems. This rebalanced coagulation system may lead to bleeding diathesis or increased clot formation. Conventional coagulation tests cannot reflect these complex changes because they can only illustrate deficiencies in the procoagulant system. Viscoelastic tests such as rotational thromboelastometry (ROTEM) have been used in LT and have shown useful for detecting coagulopathy and guiding transfusions. Implementation of ROTEM-guided bleeding management algorithms has proven effectiveness in reducing bleeding, transfusion needs, complication rates, and healthcare costs in LT. This document is intended to provide a practice algorithm for the management of major bleeding and coagulopathy during LT and to encourage adaptation of the guidelines to individual institutional circumstances and resources.

Acute pancreatitis induces a transient hypercoagulable state in murine models

Background/ObjectivesAlthough understudied, risk of venous thromboembolism (VTE) appears to be increased during acute pancreatitis (AP). We aimed to further characterize a hypercoagulable state associated with AP utilizing thromboelastography (TEG), a readily available, point of care test. MethodsAP was induced in C57/Bl6 mice using l-arginine and caerulein. TEG was performed with citrated native samples. The maximum amplitude (MA) and coagulation index (CI), a composite marker of coagulability, were evaluated. Platelet aggregation was assessed using whole blood collagen-activated platelet impedance aggregometry. Circulating tissue factor (TF), the initiator of extrinsic coagulation, was measured with ELISA. A VTE model using IVC ligation followed by measurement of clot size and weight was evaluated. After IRB approval and consent, blood samples from patients hospitalized with a diagnosis of AP were evaluated by TEG. ResultsMice with AP displayed a significant increase in MA and CI, consistent with hypercoagulability. Hypercoagulability peaked at 24 h after induction of pancreatitis, then returned to baseline by 72 h. AP resulted in significantly increased platelet aggregation and elevated circulating TF. Increased clot formation with AP was observed in an in vivo model of deep vein thrombosis. In a proof of concept, correlative study, over two thirds of patients with AP demonstrated an elevated MA and CI compared to the normal range, consistent with hypercoagulability. ConclusionsMurine acute pancreatitis results in a transient hypercoagulable state that can be assessed by TEG. Correlative evidence for hypercoagulability was also demonstrated in human pancreatitis. Further study to correlate coagulation measures to incidence of VTE in AP is warranted.

Only Subclinical Alterations in the Haemostatic System of People with Diabetes after COVID-19 Vaccination.

People with diabetes have an increased risk of experiencing adverse COVID-19 outcomes. COVID-19 vaccination is, therefore, highly recommended. However, people with diabetes have an inherently elevated risk of thrombotic events and the impact of the vaccination on the coagulation system in this patient population remains to be elucidated. The aim of this study was to investigate the impact of COVID-19 vaccination on the haemostatic system in people with type 1 or type 2 diabetes. We evaluated the effects of COVID-19 vaccination (BioNTech Pfizer, Moderna, AstraZeneca) on standard coagulation parameters, whole blood coagulation (Thrombelastometry), platelet function (impedance aggregation), and thrombin generation (calibrated automated thrombography) in people with type 1 diabetes mellitus (n = 41) and type 2 diabetes mellitus (n = 37). Blood sampling points were prior to vaccination and two weeks after the respective vaccination. Thrombelastometry measurements indicated moderately increased clot formation post-vaccination in people with type 1, as well as with type 2, diabetes: "Clot formation times" were significantly shorter, and both "maximum clot firmness" and "alpha angles" were significantly higher, as compared to the respective pre-vaccination values. Therefore, TEM parameters were not altered after vaccination in patients receiving ASA. Moreover, platelet aggregation was enhanced in people with type 1 diabetes, and plasma levels of D-Dimer were increased in people with type 2 diabetes, following COVID-19 vaccination. All other standard coagulation parameters, as well as thrombin generation, were not affected by the vaccination. The coagulation responses of people with diabetes to COVID-19 vaccination were only subclinical and comparable to those observed in healthy individuals. Our findings suggest that people with diabetes do not face an increased activation of the coagulation post-vaccination.

Hyperfibrinolysis drives mechanical instabilities in a simulated model of trauma induced coagulopathy

IntroductionTrauma induced coagulopathy (TIC) is common after severe trauma, increasing transfusion requirements and mortality among patients. TIC has several phenotypes, with primary hyperfibrinolysis being among the most lethal. We aimed to investigate the contribution of hypercoagulation, hemodilution, and fibrinolytic activation to the hyperfibrinolytic phenotype of TIC, by examining fibrin formation in a plasma-based model of TIC. We hypothesized that instabilities arising from TIC will be due primarily to increased fibrinolytic activation rather than hemodilution or tissue factor (TF) induced hypercoagulation. MethodsThe influence of TF, hemodilution, fibrinogen consumption, tissue plasminogen activator (tPA), and the antifibrinolytic tranexamic acid (TXA) on plasma clot formation and structure were examined using rheometry, optical properties, and confocal microscopy. These were then compared to plasma samples from trauma patients at risk of developing TIC. ResultsCombining TF-induced clot formation, 15 % hemodilution, fibrinogen consumption, and tPA-induced fibrinolysis, the clot characteristics and hyperfibrinolysis were consistent with primary hyperfibrinolysis. TF primarily increased fibrin polymerization rates and reduced fiber length. Hemodilution decreased clot optical density but had no significant effect on mechanical clot stiffness. TPA addition induced primary clot lysis as observed mechanically and optically. TXA restored mechanical clot formation but did not restore clot structure to control levels. Patients at risk of TIC showed increased clot formation, and lysis like that of our simulated model. ConclusionsThis simulated TIC plasma model demonstrated that fibrinolytic activation is a primary driver of instability during TIC and that clot mechanics can be restored, but clot structure remains altered with TXA treatment.

S1932 Acute Pancreatitis After Liver Biopsy: Clots or Stones?

Introduction: Percutaneous liver biopsy is a frequently utilized tool in the diagnosis of many liver diseases including metastatic liver lesions. Bleeding complications occur in 1% of cases and hemobilia is rare. Hemobilia causing acute pancreatitis after percutaneous liver biopsy is extremely rare with approximately 20 reported cases in literature. Symptoms include right upper quadrant pain, nausea, jaundice, hematemesis, hematochezia or melena. We present an interesting case of a woman who developed acute pancreatitis secondary to hemobilia after liver biopsy. Case Description/Methods: A 60-year-old female with recurrent high-grade serious peritoneal cancer and recent metastases to liver was admitted for acute pancreatitis. Patient presented with nausea and right upper quadrant pain that started after percutaneous liver biopsy one day prior. Labs notable for Hgb 10.4, lipase > 3000, amylase 3349, total bilirubin 3.6, direct 3.3, AST 631, ALT 482, alkaline phosphatase 240. CT A/P showed diffuse swelling of pancreas with fluid and mesenteric fat stranding suspicious for acute pancreatitis. Given liver enzyme elevation with mixed hepatocellular and cholestasis pattern, abdominal MRI was obtained to evaluate any biliary obstruction. MRI showed mildly prominent CBD at 0.8 cm with debris in distal CBD, cystic duct, and gallbladder indicative of hemorrhagic products. No hematoma noted in the liver parenchyma. Patient underwent ERCP with sphincterotomy and clots were cleared from the duct with balloon sweep (Figure). Discussion: Hemobilia, described as upper GI bleeding within the biliary tree has a reported incidence of less than 0.059% after percutaneous liver biopsy. It can occur within a few hours or several days later, with an average interval of five days. Although rare, hemobilia can lead to acute pancreatitis in a mechanism similar to gallstone pancreatitis. Excessive bleeding and increased clot formation cause obstruction in the biliary tree and impedes drainage from the ampulla which in turn triggers pancreatitis. Diagnosis can be made by ultrasound, angiography, ERCP, or MRCP. Treatment depends on the extent of bleeding and includes conservative management to advanced interventions-hepatic angiography with arterial embolization, ERCP with sphincterotomy or stent placement. Thus, our case highlights importance of recognizing hemobilia as an etiology for acute pancreatitis after percutaneous liver biopsy.Figure 1.: Removal of blood clots from bile duct by balloon sweep via ERCP

Computational modeling of hypercoagulability in COVID-19

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100 million people worldwide and claimed millions of lives. While the leading cause of mortality in COVID-19 patients is the hypoxic respiratory failure from acute respiratory distress syndrome, there is accumulating evidence that shows excessive coagulation also increases the fatalities in COVID-19. Thus, there is a pressing demand to understand the association between COVID-19-induced hypercoagulability and the extent of formation of undesired blood clots. Mathematical modeling of coagulation has been used as an important tool to identify novel reaction mechanisms and to identify targets for new drugs. Here, we employ the coagulation factor data of COVID-19 patients reported from published studies as inputs for two mathematical models of coagulation to identify how the concentrations of coagulation factors change in these patients. Our simulation results show that while the levels of many of the abnormal coagulation factors measured in COVID-19 patients promote the generation of thrombin and fibrin, two key components of blood clots, the increased level of fibrinogen and then the reduced level of antithrombin are the factors most responsible for boosting the level of fibrin and thrombin, respectively. Altogether, our study demonstrates the potential of mathematical modeling to identify coagulation factors responsible for the increased clot formation in COVID-19 patients where clinical data is scarce.

Inversion of Left Atrial Appendage Will Cause Compressive Stresses in the Tissue: Simulation Study of Potential Therapy

In atrial fibrillation (AF), thromboembolic events can result from the particular conformation of the left atrial appendage (LAA) bearing increased clot formation and accumulation. Current therapies to reduce the risk of adverse events rely on surgical exclusion or percutaneous occlusion, each of which has drawbacks limiting application and efficacy. We sought to quantify the hemodynamic and structural loads of a novel potential procedure to partially invert the “dead” LAA space to eliminate the auricle apex where clots develop. A realistic left atrial geometry was first achieved from the heart anatomy of the Living Heart Human Model (LHHM) and then the left atrial appendage inversion (LAAI) was simulated by finite-element analysis. The LAAI procedure was simulated by pulling the elements at the LAA tip and prescribing a displacement motion along a predefined path. The deformed configuration was then used to develop a computational flow analysis of LAAI. Results demonstrated that the inverted LAA wall undergoes a change in the stress distribution from tensile to compressive in the inverted appendage, and this can lead to resorption of the LAA tissue as per a reduced stress/resorption relationship. Computational flow analyses highlighted a slightly nested low-flow velocity pattern for the inverted LAA with minimal differences from that of a model without inversion of the LAA apex. Our study revealed important insights into the biomechanics of LAAI and demonstrated the inversion of the stress field (from tensile to compressive), which &can ultimately lead the long-term resorption of the LAA.

Donor and Storage Duration Impact Cold-Stored Platelet Function More Than Handling Protocols

Cold storage of platelets has the potential to mitigate the logistical and financial burdens associated with platelet inventory management. In addition, cold-stored platelets (CS-PLT, stored at 2-6°C) have better preserved hemostatic function than room temperature (RT)-stored platelets (RT-PLT), suggesting that CS-PLT may provide improved hemostatic resuscitation in actively bleeding patients as compared to RT-PLT when transfused. With CS-PLT clinical use on the rise, questions regarding CS-PLT inventory management have been raised - specifically how should CS-PLT be stored and handled over the course of storage (after collection and prior to transfusion). RT-PLT are currently stored flat on specialized porous racks and agitated at 18-22°C, which allows for oxygenation. These products are stored out to 5 days, with 7 days allowed for large volume delayed sampling collections. In contrast, refrigerated blood products (i.e., packed red blood cells and whole blood) are stored vertically in pull out drawers, with the goal of reducing harmful oxidizing damage to red blood cells. Notably, refrigeration is known to induce aggregation of platelets, a feature historically associated with bacterial contamination in RT-PLT. In order to diminish concerns regarding aggregate formation, it may be beneficial to massage CS-PLT briefly each day to reduce aggregate formation, yet how this may impact hemostatic function is unknown. To this end, optimal storage and handling conditions for CS-PLT remain to be determined.The objective of our study was to measure CS-PLT hemostatic function in response to two major storage and handling variables: agitation and massage to reduce aggregation in the bag. Single donor apheresis platelet units (single Trima collection in plasma, split equally into two bags to control for donor variability; n=20 donors in 40 bags) were purchased from our regional blood center and delivered to our lab on day of collection. Upon arrival, units were spiked and sampled under sterile conditions for baseline (day 0) profiling, assigned to one of the study groups, and stored accordingly. Our study groups were as follows: “Agitated” (n=10 units), “Not Agitated” (n=10 units, paired with Agitated donors), “Massaged Daily”, (massaged 60s daily, n=10 units), and “Massaged at Sampling” (massaged only at sampling, n=10 units, paired with Massaged Daily donors). “Agitated” platelet units were stored in a custom refrigerated shaker (courtesy Helmer Scientific). For the other groups, platelet units were stored in a walk-in cold room. All units were stored on the same style perforated agitator rack, with agitation either powered on or off as assigned. Units from all groups were sampled under sterile conditions at days 2, 5, 7, 14, and 21 of storage, using 8 mL draws at each time point to ensure equal volume removal over the course of storage. Hemostatic function was assayed using light transmission aggregometry (LTA; ADP, collagen, epinephrine agonists), rotational thromboelastometry (ROTEM; ExTEM, InTEM agonists), and thrombin generation in response to 5 pM tissue factor. Platelet counts (x10 3/µL) were obtained using a hematology analyzer.During the first week of storage, there were no significant differences in the hemostatic profiles between study groups. While platelet counts, endogenous thrombin potential, and ROTEM clot formation time and maximum clot firmness were fairly stable over the first 7 days of storage across all groups, there was a > 50% reduction from baseline in aggregation responses to both ADP and collagen across all 4 groups, suggesting that platelet aggregation, as detected by LTA, may not be the best representative of CS-PLT function. By day 21 of storage, there was a robust increase in endogenous thrombin potential in all study groups when compared to baseline (Agitated, 22%; Not Agitated, 70%; Massaged Daily, 51%; Massaged at Sampling, 47%). Despite this increase in thrombin generation, after extrinsic activation day 21 CS-PLT in all groups had two-fold increased clot formation times compared to baseline (Agitated, 185%; Not Agitated, 223%; Massaged Daily, 236%; Massaged at Sampling, 190%), and reduced maximum clot firmness compared to baseline (Agitated, -44%; Not Agitated, -36%; Massaged Daily, -54%; Massaged at Sampling, -43%). These data suggest that storage duration, and not agitation nor massaging to reduce aggregates, has the most impact on CS-PLT hemostatic function. DisclosuresSpinella: Cerus Corporation: Consultancy, Research Funding; Secure Transfusion Services: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Coagulation profiles and viscoelastic testing in multisystem inflammatory syndrome in children.

To characterize viscoelastic testing profiles of children with multisystem inflammatory syndrome in children (MIS-C). This single-center retrospective review included 30 patients diagnosed with MIS-C from March 1 to September 1, 2020. Thromboelastography (TEG) with platelet mapping was performed in 19 (63%) patients and compared to age- and sex-matched controls prior to cardiac surgery. Relationships between TEG parameters and inflammatory markers were assessed using correlation. Patients with MIS-C had abnormal TEG results compared to controls, including decreased kinetic (K) time (1.1 vs. 1.7minutes, p<.01), increased alpha angle (75.0° vs. 65.7°, p<.01), increased maximum amplitude (70.8 vs. 58.3mm, p<.01), and decreased lysis in 30minutes (Ly30) (1.1% vs. 3.7%, p=.03); consistent with increased clot formation rate and strength, and reduced fibrinolysis. TEG maximum amplitude was moderately correlated with erythrocyte sedimentation rate (ESR) (r=0.60, p=.02), initial platelet count (r=0.67, p<.01), and peak platelet count (r=0.51, p=.03). TEG alpha angle was moderately correlated with peak platelet count (r=0.54, p=.02). Seventeen (57%) patients received aspirin (ASA) and anticoagulation, five (17%) received only ASA, and three (10%) received only anticoagulation. No patients had a symptomatic thrombotic event. Six (20%) patients had a bleeding event, none of which was major. Patients with MIS-C had evidence of hypercoagulability on TEG. Increased ESR and platelets were associated with higher clot strength. Patients were prophylactically treated with ASA or anticoagulation with no symptomatic thrombosis or major bleeding. Further multicenter study is required to characterize the rate of thrombosis and optimal thromboprophylaxis algorithm in this patient population.

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Coagulation, hemostasis, and transfusion during liver transplantation.

Liver disease is associated with complex disturbances of hemostasis that affect both the pro- and anticoagulant systems. This results in a "rebalanced" coagulation system that may result in bleeding diathesis or increased clot formation. Conventional coagulation models of coagulation such as the waterfall cascade model and conventional coagulation tests are not able to reflect these complex changes as they only account for deficiencies of the pro-coagulant system. Viscoelastic tests such as rotational thromboelastometry or thromboelastography may be more suitable to assess coagulation and guide transfusions in liver transplants. Specific recommendations for transfusion of platelets, fresh frozen plasma, cryoprecipitate, and fibrinogen as well as factor concentrates are discussed. In general transfusion should not only be guided by laboratory values but it also includes a clinical assessment of clot formation.

Detection of the State of Thrombotic Readiness in Rats under One-Time Suprathreshold Physical Activity of Various Durations by Means of Thromboelastography

Suprathreshold physical activity causing distress in the organism can lead to the damage of various organs and systems including the hemostatic system. A modern integrated method of the hemostatic system assessment is thromboelastography. The purpose of the present study was to evaluate the state of the hemostatic system under one-time suprathreshold physical activity of various durations by means of thromboelastography. Experimental groups of rats were exposed to 4-hour and 8-hour physical activities in the form of forced running on a moving platform with the speed of 6-8 m/min. Immediately after the one-time physical activity, blood samples taken from rats were examined using the thrombelastograph in the Natem mode for 35 minutes. The 4-hour physical activity caused a reduction in coagulation time (CT) and an increase in the alpha angle and the maximum clot firmness (MCF). After the 8-hour activity, the thrombelastograph registered a reduction in coagulation time (CT), an increase in the alpha angle, a decrease in the clot formation time (CFT), a decrease in the maximum clot firmness (MCF), and a reduction in the maximum clot lysis (ML). The 4-hour physical activity resulted in partial activation of the hemostatic system without changing the fibrinolytic activity of blood plasm. The changes revealed in thromboelastography parameters indicate a high risk of the development of thrombotic readiness. The 8-hour physical activity causes a shift of the hemostatic system parameters in rats towards the increased clot formation: hypercoagulation, fibrinogen and platelet consumption, inhibition of fibrinolysis. The combination of changes in thromboelastogram parameters is indicative of the development of thrombotic readiness

Ex vivo properties of plasma clot formation and lysis in patients with cancer at risk for venous thromboembolism, arterial thrombosis, and death

A prothrombotic state is frequently observed in patients with cancer and contributes to the risks of venous thromboembolism (VTE), arterial thromboembolism (ATE), tumor progression, and death. Altered ex vivo properties of plasma clot formation and lysis have been observed in patients with cancer. The aim of this prospective study was to comprehensively characterize the relationship between plasma clot properties, inflammation, hypercoagulability, thrombotic complications, and mortality in patients with cancer using a tissue-factor-based turbidimetric assay of clot formation and lysis. Turbidity parameters were determined in 815 patients with newly-diagnosed or recurrent cancer and 97 healthy controls. Patients were followed-up for 2 years and rates of VTE (n = 72 events), ATE (n = 21 events), and death (n = 304 events) were assessed. Compared to controls, cancer patients' turbidity profiles showed an increased clot formation potential and higher resistance toward fibrinolysis. Elevated biomarkers of inflammation and hemostasis, such as C-reactive protein, FVIII, and thrombin generation explained substantial amounts of variation in turbidity parameters. In a prospective analysis, altered parameters of clot formation identified cancer patients at high risk of ATE (Hazard ratio [HR] per doubling of peak absorbance: 4.43, 95% CI: 1.50-13.07, P = 0.007) and death (HR per doubling of peak absorbance: 2.73, 2.00-3.72, P< 0.0001); these findings were independent of other prognostic covariates. Contrarily, turbidity parameters were not associated with risk of VTE (HR per doubling of peak absorbance: 1.15, 0.66-2.01, P = 0.62). We conclude that patients with cancer have altered ex vivo properties of clot formation which predict risks of ATE and mortality but not VTE.

Rotational thromboelastometry (ROTEM) 24 hours post liver transplantation predicts early allograft dysfunction.

One hundred sixty-four patients who underwent LT were prospectively included in the present study. Patient demographics, intraoperative blood loss and transfusion were recorded at the time of LT. Lactate levels were recorded during surgery and daily for the first 3 postoperative days. Standard and derived rotational thromboelastometry (ROTEM) parameters were recorded 24 hours after LT. EAD was diagnosed according to Nanashima criteria and post anaesthesia care unit length of stay was recorded. Forty-seven patients (28.6%) developed EAD. Intraoperative blood loss (p = 0.01), packed red blood cells (p = 0.04) and fresh frozen plasma (p = 0.01) transfusion represented intraoperative risk factors for EAD. Lactate levels were significantly higher in patients with EAD at all time points. Patients with EAD demonstrated an increased clot formation time and decreased maximum clot firmness in both intrinsically (p < 0.01) and extrinsically (p < 0.01) activated assay, a decreased thrombin potential index (p < 0.01), area under the curve (p < 0.01) and clot elasticity (p < 0.01) on ROTEM assay. Our results show that both standard and derived ROTEM parameters may indicate early signs of graft failure and can aid in the diagnosis of EAD.

MASP-1 of the complement system enhances clot formation in a microvascular whole blood flow model.

The complement and coagulation systems closely interact with each other. These interactions are believed to contribute to the proinflammatory and prothrombotic environment involved in the development of thrombotic complications in many diseases. Complement MASP-1 (mannan-binding lectin-associated serine protease-1) activates coagulation factors and promotes clot formation. However, this was mainly shown in purified or plasma-based static systems. Here we describe the role of MASP-1 and complement activation in fibrin clot formation in a microvascular, whole blood flow model. This microfluidic system simulates blood flow through microvessels at physiological flow and shear rates and represents the closest model system to human physiology so far. It features parallel microchannels cultured with endothelial cells in a transparent microfluidic chip allowing real-time evaluation of clot formation by confocal microscopy. To test their effects on clot formation, we added the following activators or inhibitors (individually or in combination) to whole blood and performed perfusion experiments: rMASP-1cf (recombinant active form of MASP-1), complement activator zymosan, selective MASP-1 inhibitor SGMI-1 (based on the Schistocerca gregaria protease inhibitor scaffold), classical pathway inhibitor rSALO (recombinant salivary anti-complement from Lutzomyia longipalpis). Addition of rMASP-1cf resulted in accelerated fibrin clot formation while addition of SGMI-1 delayed it. Complement activation by zymosan led to increased clot formation and this effect was partially reversed by addition of rSALO and almost abolished in combination with SGMI-1. We show for the first time a strong influence of MASP-1, complement activation and pathway-specific inhibition on coagulation in a microvascular flow system that is closest to human physiology, further underpinning the in vivo relevance of coagulation and complement interactions.

Selection of Tissue Factor-Deficient Cell Transplants as a Novel Strategy for Improving Hemocompatibility of Human Bone Marrow Stromal Cells.

Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation.Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC.Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells.Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.

Evaluation of the hemocompatibility of RADA 16-I peptide.

RADA 16-I is an ionic self-assembling peptide that can form macroscopic scaffolds through β-sheet structures which are used in favor of cell growth and tissue engineering. This peptide has also the ability to stop bleeding effectively and quickly (∼20 seconds) when applied directly to the injuries. This study is focused on coagulation process, platelet aggregation, C3 and C4 concentrations, CBC counting, hemolysis, and white blood cell morphology tests to analyze hemocompatibility of RADA 16-I at different concentrations - 0.1, 0.2, 0.3 and 0.5%. According to the results, RADA 16-I hydrogel decreased the number of blood cells, slightly increased clot formation time and platelet aggregation, and yielded negligible hemolysis and only small changes in C3 and C4 concentrations and white blood cell morphology. All by all, the in vitro tests of hemocompatibility showed no perturbation in the blood composition when the peptides were in contact with the blood. The observed rapid hemostasis might be a result of increasing local concentrations of molecules involved in the formation of clot near the peptide hydrogel, thereby making a barrier which ended up with complete hemostasis. In conclusion, our experiments strongly supported further development of biomaterials based on RADA 16-I peptide.

Hypercoagulability progresses to hypocoagulability during evolution of acetaminophen-induced acute liver injury in pigs

Increases in prothrombin time (PT) and international normalised ratio (INR) characterise acute liver injury (ALI) and failure (ALF), yet a wide heterogeneity in clotting abnormalities exists. This study defines evolution of coagulopathy in 10 pigs with acetaminophen (APAP)-induced ALI compared to 3 Controls. APAP administration began at 0 h and continued to ‘ALF’, defined as INR >3. In APAP pigs, INR was 1.05 ± 0.02 at 0 h, 2.15 ± 0.43 at 16 h and > 3 at 18 ± 1 h. At 12 h thromboelastography (TEG) demonstrated increased clot formation rate, associated with portal vein platelet aggregates and reductions in protein C, protein S, antithrombin and A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats–13 (ADAMTS-13) to 60%, 24%, 47% and 32% normal respectively. At 18 ± 1 h, INR > 3 was associated with: hypocoagulable TEG profile with heparin-like effect; falls in thrombin generation, Factor V and Factor VIII to 52%, 19% and 17% normal respectively; further decline in anticoagulants; thrombocytopenia; neutrophilia and endotoxemia. Multivariate analysis, found that ADAMTS-13 was an independent predictor of a hypercoagulable TEG profile and platelet count, endotoxin, Protein C and fibrinogen were independent predictors of a hypocoagulable TEG profile. INR remained normal in Controls. Dynamic changes in coagulation occur with progression of ALI: a pro-thrombotic state progresses to hypocoagulability.

Increased Clot Formation in the Absence of Increased Thrombin Generation in Patients with Peripheral Arterial Disease: A Case-Control Study.

In peripheral arterial disease (PAD), activation of the hemostatic system may contribute to atherosclerosis progression and atherothrombotic events. This case-control study assesses the overall coagulation status in PAD patients by evaluating coagulation markers in combination with thrombin generation potential, whole blood (WB) clot formation, and fibrinolysis. In blood from 40 PAD patients (n = 20 with cardiovascular event within 1 year after initial diagnosis, n = 20 without) and 40 apparently healthy controls, thrombin generation was determined in WB and platelet-poor plasma. Whole blood rotational thromboelastometry (ROTEM) measurements were triggered with tissue factor with/without tissue plasminogen activator. We observed increased levels of erythrocyte sedimentation rate, leukocytes, eosinophil granulocytes, vWF antigen, fibrinogen, and D-dimer in PAD patients (p < 0.05). Markers of thrombin generation potential showed no difference between patients and healthy controls. In PAD patients with event compared to patients without, WB-thrombin generation showed a lower thrombin potential when triggered with 0 and 2.5 pM tissue factor. The ROTEM clotting assay showed significantly faster clot formation and increased clot firmness in PAD patients compared to controls. No significant differences were found for parameters of clot degradation. There are no significant differences between the thrombin generation profiles of PAD patients and healthy controls. Between PAD patients with and without cardiovascular event, the WB thrombin generation appears to differ. Mechanistically, PAD patients show an increased ability to form a stable clot in WB in comparison to healthy controls. This is most likely due to the increased fibrinogen levels related to the inflammation in atherosclerosis, confirming the importance of the inflammation-coagulation axis.