Comparison of viscoelastic test results from blood collected near simultaneously from the jugular and saphenous veins in cats

Objectives The aim of this study was to compare viscoelastic test results from samples collected from a jugular vein using a 20 G needle and a medial saphenous vein using a 22 G needle in cats presenting for elective ovariohysterectomy (OHE) or castration. Methods Forty apparently healthy cats (20 males and 20 females) presenting for elective OHE or castration were included in a prospective study observing viscoelastic test results from central and peripherally collected whole blood. Cats were anesthetized during blood collection with a standardized protocol including buprenorphine, ketamine, dexmedetomidine and isoflurane. Blood samples from jugular and saphenous veins were collected near simultaneously. Viscoelastic evaluations of whole blood were performed using a point-of-care device measuring clot time (CT), clot formation time (CFT), alpha angle (α), maximum clot formation (MCF), and amplitude at 10 and 20 mins (A10 and A20, respectively). Viscoelastometry continued post-clot time to determine a lysis index at 30 and 45 mins (LI30 and LI45, respectively) to assess fibrinolysis. Results Studied cats had a median age of 18 months (range 5 months to 5 years) and a median weight of 3.6 kg (range 2.7–5.9 kg). A total of 80 samples were available for analysis. While lysis indices were not different, viscoelastic measures of coagulation differed between sampling sites (CT, P <0.005; CFT, P = 0.01; α, P <0.05; MCF, P <0.0005; A10, P <0.0005; A20, P <0.0005). Conclusions and relevance Viscoelastic results from jugular venous blood samples appear to be more hypercoagulable than those collected from the medial saphenous vein, suggesting that the same site should be used consistently for serial monitoring or for collecting study data.

Evaluation of a Next-Generation FVIII Mimetic Antibody, Mim8, in Combination with Recombinant FVIIa, FVIII and Activated Prothrombin Complex Concentrate

Introduction: Coagulation Factor VIII (FVIII) mimetics is a new class of molecules restoring the hemostatic capacity in blood lacking FVIII irrespective of the presence of inhibitory anti-FVIII antibodies. FVIII mimetics are bispecific antibodies bridging FIXa and FX on platelet surface, enhancing FX activation and thereby coagulation. Despite a significant reported prophylactic effect of the FVIII-mimetic emicizumab (Hemlibra®) in persons with Hemophilia A with and without inhibitors [Oldenburg et al., NEJM, 2017; Mahlangu et al., NEJM, 2018], bleeding may still occur during treatment, requiring intervention with FVIII or bypassing agents, i.e. recombinant FVIIa (rFVIIa) or activated prothrombin complex concentrate (aPCC). The combined treatment with emicizumab and aPCC has under certain conditions led to thrombotic events and microangiopathies in the treated patients, whereas the combination with rFVIIa has been safe [Levy et al., J Thromb Haemost, 2019]. We have developed a novel, next-generation FVIII mimetic antibody, Mim8, that potently enhances coagulation in pre-clinical models [manuscript in preparation]. We evaluated the combination of Mim8 with recombinant FVIII (rFVIII), rFVIIa, and aPCC by thrombin generation assay (TGA) and by thromboelastography (TEG), in haemophilia A condition. Material and methods: Mim8 was tested in a range including the expected therapeutic concentration (10 - 50 nM, based on pre-clinical data) and studied in parallel with a sequence identical analogue (SIA) of emicizumab (concentrations including the therapeutic range of ⁓350 nM [Oldenburg et al., NEJM, 2017]). The compounds were tested in combinations with rFVIII, rFVIIa or aPCC. For TGA, coagulation was initiated with addition of FXIa or tissue factor (TF), and peak thrombin is reported. For TEG, haemophilia A was induced in whole blood from healthy volunteers by adding an anti-FVIII polyclonal antibody, and clot formation initiated by TF. The clot time (R-time) and clot development (alpha-angle) are reported. Under the chosen experimental conditions, TEG is particularly useful for studying the interaction between Mim8 and rFVIIa as clinically relevant doses of rFVIIa (i.e. 25 and 75 nM) can normalize haemophilic blood in TEG, while a very poor response is observed in TGA. Results: In TGA, Mim8 alone induced a concentration-dependent increase in thrombin generation with both triggers (TF or FXIa). In combination with rFVIII, Mim8 generated thrombin levels within normal range and did not inhibit the effect of 1 IU/mL rFVIII. The combined addition of aPCC 1 IU/mL and Mim8 generated TF-triggered thrombin peaks up to thirteen-fold higher than in normal plasma, suggesting synergistic effect between Mim8 and aPCC. Similar data were obtained when aPCC and emicizumab SIA were combined. When triggered by FXIa, the thrombin peaks generated by the combination of aPCC 1 IU/mL and Mim8 were also above normal plasma values. In contrast, the combination of rFVIIa (up to 75 nM) and Mim8 (up to 50 nM) showed an additive effect in TF-triggered assays i.e. the thrombin peak did not increase more than the individual contribution from each molecule. In TEG, Mim8 alone normalized clot time of haemophilia A blood. When rFVIIa (up to 75 nM) was combined with Mim8, clot time stayed within normal range. In addition, clot time induced by up to 50 nM of Mim8 in combination with 75 nM rFVIIa was still significantly longer than emicizumab SIA 350 nM + 1 IU/mL aPCC (P < 0.01). Also, the combination of rFVIIa and Mim8 at any concentrations tested only increased clot development, or alpha-angle, up to normal blood levels. In contrast, the addition of only 0.25 IU/mL aPCC combined with 5 nM of Mim8, or only 0.1 IU/mL aPCC combined with 50 nM Mim8, decreased the clot time below normal value. Conclusions: Using TGA and TEG, Mim8, at the anticipated clinical concentrations, did not interfere with the effect of rFVIII, i.e. only an additive effect of Mim8 and rFVIII was observed. Similarly, rFVIIa had an additive effect with Mim8. However, as expected, aPCC demonstrated a synergistic effect when combined with Mim8. Clot formation parameters and thrombin peak exceeded normal levels, even at a reduced concentration of aPCC. The data obtained with Mim8 and emicizumab SIA were comparable, suggesting that the synergistic effect with aPCC is a class effect. Disclosures Burnier: Novo Nordisk A/S: Employment. Hermit:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hilden:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties. Ezban:Novo Nordisk A/S: Employment. Kjellev:Novo Nordisk A/S: Employment, Equity Ownership.

Improving mechanical thrombectomy time metrics in the angiography suite: Stroke cart, parallel workflows, and conscious sedation

Purpose Earlier reperfusion of large-vessel occlusion (LVO) stroke improves functional outcomes. We hypothesize that use of a stroke cart in the angiography suite, containing all commonly used procedural equipment in a mechanical thrombectomy, combined with parallel staff workflows, and use of conscious sedation when possible, improve mechanical thrombectomy time metrics. Methods We identified 47 consecutive LVO patients who underwent mechanical thrombectomy at our center, retrospectively and prospectively from implementation of these three workflow changes (19 pre- and 28 post-). For each patient, last known normal, NIHSS, angiography suite in-room time, type of anesthesia, groin puncture time, on-clot time, recanalization time, LVO location, number of passes, device(s) used, mTICI score, and outcome (mRS) were recorded. Between-group comparisons of time metrics and multivariate regression were performed. Results Stroke cart, parallel workflows, and primary use of conscious sedation decreased in-room time to groin puncture (−21.3 min, p < 0.0001), in-room to on-clot time (−24.1 min, p = 0.001), and in-room to reperfusion time (−29.5 min, p = 0.01). In a multivariate analysis, endotracheal intubation and general anesthesia were found to significantly increase in-room to on-clot time ( p = 0.01), in-room to reperfusion time ( p = 0.01), and groin puncture to on-clot time ( p = 0.05). The number of patients achieving a good outcome (mRS 0−2), however, did not significantly differ between the two groups (9/18 (47%) vs 14/28 (50%), p = 0.60). Conclusions Use of a stroke cart, parallel workflows by neurointerventionalists, technologists, and nursing staff, and use of conscious sedation may be useful to other institutions in efforts to improve procedural times.

Hypercoagulability in Sickle Cell Disease: The Importance of the Cellular Component of Blood

Sickle cell disease (SCD) is considered to be a hypercoagulable state with chronic activation of coagulation and an increased incidence of thrombotic events. However, there is no consensus on whether global assays of thrombin generation in platelet poor (PPP) or platelet rich (PRP) plasma display an increased thrombin generation potential in SCD (reviewed in Lim et al. Curr. Opin. Hematol. 2013). Based on our recent observation that RBC contribute to thrombin generation in whole blood (Whelihan et al. Blood 2012), we hypothesized that the cellular components in blood (notably RBCs) contribute to enhanced thrombin generation in SCD. 25 SCD patients in a non-crisis, “steady state” and 25 healthy race–matched controls were recruited for study. Whole blood thrombin generation, thromboelastography (TEG) and plasma-based thrombin generation assays (TGA) were performed on contiguous blood samples from each individual. Complete blood counts, as well as quantification of phosphatidylserine (PS) exposure by RBCs (assayed by Annexin V binding in flow cytometry) were also performed. Whole blood thrombin generation was monitored by serial α-thrombin-antithrombin (TAT) complex formation following activation by both the extrinsic pathway (5 pM recombinant tissue factor (TF)) and intrinsic (native contact activation) pathways. Results. With extrinsic activation, controls clotted on average at 3.9±0.5 min (mean±SD), generated TAT at a rate of 38.8±22.2 nM/min and reached a maximum level of 261±58nM. In patients with SCD, clot time (4.4±0.8 min, ƿ=0.04) and the maximum rate of TAT generation (41.5±19.4 nM/min, ƿ=0.65) were similar to that observed in controls while the maximum level (369±123 nM) of TAT generated was significantly higher (ƿ=0.0026). With contact activation, there was no significant difference in clot time (7.2±1.2 min vs 6.4±1.0 min, ƿ=0.07) or the maximum rate of TAT generation (52.9.5±26.9 nM/min vs 42.3 ± 12.4 nM/min, ƿ=0.57) for the SCD and control cohorts, respectively. However, similar to what was observed with extrinsic activation, SCD patients generated a significantly higher (ƿ=0.024) maximum level of TAT (352±116 nM) than controls (276±21 nM). Interestingly, the SCD cohort showed a strong positive correlation (ƿ=<0.001) between the maximum levels of TAT generated with either stimulus (extrinsic vs intrinsic). We also examined extrinsic- and intrinsic-initiated clot formation using TEG. Re-calcification of citrated blood from SCD in the presence of TF and Corn Trypsin Inhibitor (CTI) displayed similar R (8.1±0.3 min vs 8.4±0.6 min, ƿ=0.7) and MA (68±2 mm vs 65±2 mm, ƿ=0.16) values to those exhibited by controls, respectively. The -angle however, was significantly (ƿ=0.035) higher in the SCD cohort (61±3°) compared to controls (53±3°). Contact activated blood also displayed no significant difference in R time between the two groups (14.5±3 min for SCD vs 16.5±4 min for controls). On the other hand, the MA and -angle were significantly increased (ƿ=0.04 and ƿ=0.012) in SCD (62±2 mm and 41±3°) compared to the controls (51±3 mm and 26±5°), suggesting a significant increase in the overall rate and extent of clot formation as a result of contact activation. To examine thrombin generation in the absence of cellular components, PPP was activated using an identical 5pM TF stimulus. In contrast to whole blood, TGA displayed no significant differences in the peak thrombin or maximum rate of thrombin generation between the two groups. Lag time was marginally longer and time to peak thrombin generation shorter in SCD (ƿ=0.036 and ƿ=0.03, respectively). Surprisingly, a weak negative correlation (ƿ=0.15) between RBC PS expression and total TAT was present in SCD patient samples. Conclusions: While plasma-based assays exhibit no major differences in thrombin generation potential between SCD and controls, corresponding whole blood samples showed a significant increase in overall thrombin generation and clotting potential, regardless of the initiating stimulus (extrinsic or intrinsic). Interestingly, there was no significant correlation between absolute cell counts (i.e. RBCs, Retics, Neuts, PLTs) or parameters and TAT levels. Collectively, these data make a strong case for cellular involvement in the hypercoagulability observed in SCD, but a direct role for RBC PS expression in net thrombin generation is not apparent. Disclosures No relevant conflicts of interest to declare.

The mild phenotype in severe hemophilia A with Arg1781His mutation is associated with enhanced binding affinity of factor VIII for factor X

SummaryThe clinical severity in some patients with haemophilia A appears to be unrelated to the levels of factor (F)VIII activity (FVIII:C), but mechanisms are poorly understood. We have investigated a patient with a FVIII gene mutation at Arg1781 to His (R1781H) presenting with a mild phenotype despite FVIII:C of 0.9 IU/dl. Rotational thromboelastometry using the patient’s whole blood demonstrated that the clot time and clot firmness were comparable to those usually observed at FVIII:C 5–10 IU/dl. Thrombin and FXa assays using plasma samples also showed that the peak levels of thrombin formation and the initial rate of FXa generation were comparable to those observed at FVIII:C 5–10 IU/dl. The results suggested a significantly greater haemostatic potential in this individual than in those with severe phenotype. The addition of incremental amounts of FX to control plasma with FVIII:C 0.9 IU/dl in clot waveform analyses suggested that the enhanced functional tenase assembly might have been related to changes in association between FVIII and FX. To further investigate this mechanism, we prepared a stably expressed, recombinant, B-domainless FVIII R1781H mutant. Thrombin generation assays using mixtures of control plasma and FVIII revealed that the coagulation function observed with the R1781H mutant (0.9 IU/dl) was comparable to that seen with wild-type FVIII:C at ∼5 IU/dl. In addition, the R1781H mutant demonstrated an ∼1.9-fold decrease in K m for FX compared to wild type. These results indicated that relatively enhanced binding affinity of FVIII R1781H for FX appeared to moderate the severity of the haemophilia A phenotype.Note: An account of this work was presented, in part, at the 23rd Congress of the International Society of Thrombosis and Haemostasis, July 27, 2011, Kyoto, Japan.

Procoagulant Platelet Subpopulation Formation in Whole Blood: Correlation with Whole Blood Clotting Time and 5-Year Mean Bleeding Score in Individuals with Factor VIII Deficiency.

Abstract 4022 Poster Board III-958 Different clinical phenotypes are often observed among individuals with hemorrhagic or thrombotic disorders despite the presence of the same mutation and/or factor levels suggesting that additional genetic and/or environmental factors influence clinical presentation. As thrombin plays a central role in hemostasis, factors that affect an individual's ability to generate thrombin may lead to an increased risk of hemorrhage or thrombosis. The goal of this study was to assess procoagulant platelet formation in individuals with hemophilia A using a rapid, whole blood flow cytometric assay of prothrombinase complex assembly on platelets described previously. In this assay, Ca2+-dependent factor Xa binding to activated platelets is used as a marker of thrombin generating potential as it has been shown previously to correlate with platelet prothrombinase activity in a washed platelet system. Procoagulant platelet subpopulation formation in 15 men with varying degrees of factor VIII-deficiency was evaluated and compared to two independent measures of hemostatic competence: a 5-year mean bleeding score and whole blood clot time. In these individuals, the % activated platelets binding factor Xa in whole blood varied from 2.35 – 9.0% (n = 30), which is consistent with what is observed in unaffected individuals (1.5 – 41.5%, n = 136). Bleeding was scored independently by two experienced hemophilia nurses and one hematologist at Centre Hospitalier Universitaire Sainte Justine based on each individual's bleeding history, including hemarthroses, soft tissue hematoma, and annual factor VIII usage, and averaged. The 5-year mean bleeding scores in these individuals ranged from 0 – 20.4. Linear regression analyses indicated that in this population the % activated platelets binding factor Xa correlated indirectly with the 5-year mean bleeding score, where individuals with lower bleeding scores (i.e. less bleeding episodes and/or factor VIII usage) generated larger procoagulant platelet subpopulations. The time to clot formation in a tissue factor-dependent, contact pathway-suppressed whole blood clotting assay described previously, was also determined. The clot time, which was determined visually, and ranged from ∼3 – 7 min, also correlated indirectly, though less well, with the % activated platelets binding factor Xa in whole blood (r = -0.23). Thus, consistent with what was observed with the bleeding score, those individuals who clotted more quickly (i.e. exhibited a greater degree of hemostatic competence) generated more proacoagulant platelets. Platelet procoagulant subpopulation formation was also compared to other hematological measures. An inverse correlation was observed between the % activated platelets binding factor Xa and mean platelet volume (r = -0.347), suggesting that in this population, individuals with smaller platelets (i.e. platelets with fewer prothrombinase binding sites) may generate a greater number of platelets capable of assembling prothrombinase. In contrast, no correlation was observed between whole blood platelet number and % activated platelets binding factor Xa. Interestingly, factor Xa binding was also negatively correlated with plasma levels of factor IX (r = -0.56) and factor V (r = -0.62). Platelet subpopulation formation was also weakly and negatively associated with the aPTT (r = - 0.28). In this small population of individuals with hemophilia A, whole blood platelet factor Xa binding correlates with bleeding phenotype. These observations support the notion that this measurement can be used to predict an individuals propensity to bleed or clot. Disclosures: No relevant conflicts of interest to declare.

Polyphosphate Shortens the Clotting Time of Hemophilic and Anticoagulated Plasma.

Introduction: Inorganic polyphosphate (polyP) is a negatively charged polymer of phosphate units linked by high energy phosphoanhydride bonds. Dense granules of human platelets contain abundant polyP which is released in response to thrombin stimulation. We recently reported that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway, promoting factor V activation, and abrogating tissue factor pathway inhibitor function. We now report that polyP shortens the clot time of hemophilic plasmas and plasmas containing a variety of anticoagulant drugs. Methods: Citrated plasmas were obtained from normal donors, patients receiving coumadin, and individuals with severe hemophilia A or B. In some experiments, clinically relevant concentrations of unfractionated heparin, enoxaparin, or argatroban were added to normal plasma. In other experiments, therapeutic concentrations of FIX, FVIII, or FVIIa were added to hemophilic plasma. Clotting was initiated by dilute thromboplastin. Results: Adding 33 uM polyP to normal plasma shortened clot times at all doses of heparin (by 37–55%), enoxaparin (by 35–57%), and argatroban (by 28–59%; see Figure). PolyP shortened clot times of plasmas from coumadin patients (by 29–39%) with INR values ranging from 1.7 to 4.8. PolyP also shortened the clot times of plasma from patients with hemophilia A (by 54–75%) and B (by 51–79%). PolyP alone shortened clot times of hemophilia A or B plasma equivalent to adding 6 to 15 nM recombinant FVIIa, and the polyP effect was additive to that of FVIIa, FVIII or FIX. Conclusions: PolyP shortened the in vitro clot time for all plasmas tested and was additive with replacement of missing clotting factors or recombinant FVIIa for hemophilic plasmas. These results suggest that treatment with polyP may be useful for reversal of anticoagulation in patients treated for thrombophilia, or for treatment of bleeding episodes in patients with hemophilia. Furthermore, polyP could potentially reduce the dose of replacement clotting factors or recombinant FVIIa needed to treat bleeding episodes in hemophilia. Figure Figure

Volutrauma activates the clotting cascade in the newborn but not adult rat

Coagulopathy and alveolar fibrin deposition are common in sick neonates and attributed to the primary disease, as opposed to their ventilatory support. Hypothesizing that high tidal volume ventilation activates the extrinsic coagulation pathway, we air ventilated newborn and adult rats at low (10 ml/kg) or high (30 ml/kg) tidal volume and compared them with age-matched nonventilated controls. Blood was collected at the end of the experiment for measurement of clot time, tissue factor, and other coagulation factor content. Similar measurements were obtained from lung lavage material. The newborn clot time (44 ± 1) was lower and plasma tissue factor content higher (103.4 ± 0.4) than adults (88 ± 4 s and 26.6 ± 1.4 units; P < 0.01). High, but not low, tidal volume ventilation of newborns for as little as 15 min significantly reduced clot time and increased plasma tissue factor content ( P < 0.01). High volume ventilation increased plasma factor Xa (0.1 ± 0.1 to 1.6 ± 0.4 nM; P < 0.01) and thrombin (1.3 ± 0.2 to 2.2 ± 0.4 nM; P < 0.05) and decreased antithrombin (0.12 ± 0.01 to 0.05 ± 0.01; P < 0.01) in the newborn. Lung lavage material of high volume-ventilated newborns showed increased ( P < 0.01) factor Xa and thrombin. No changes in these parameters were observed in adult rats that were high volume ventilated for up to 90 min. Compared with adults, newborn rats have a greater propensity for volutrauma-activated intravascular coagulation. These data suggest that mechanical ventilation promotes neonatal thrombosis via lung tissue factor release.

Hemophilia and the Dynamics of Hemostasis.

The conversion of fibrinogen to fibrin and cross-linking to form a stable clot are key processes in effective hemostasis. We investigated fibrinopeptide (FP) A and FPB release, factor XIII (FXIII) activation and fibrin mass in tissue factor initiated coagulation in whole blood from individuals with hemophilia (n=8) and healthy subjects (n=35). For hemophilia whole blood, the rates and patterns of fibrin formation are altered when compared to blood from healthy individuals. The rate of FPA release is decreased from 2.3μ M/min in healthy individuals to 1.5μ M/min in hemophilia and more significantly the rate of FPB release is decreased from 1.3μ M/min to 0.19 μ M/min in hemophilia whole blood. The activated form of FXIII in hemophilia whole blood is formed at a rate of 4nM/min versus healthy individuals where the rate is accelerated to 7nM/min. Prior to clot time in hemophilia whole blood, FPB release is reduced. Whereas more FPA is released in hemophilia whole blood prior to clot time than in whole blood from healthy individuals, 67% versus 47%, respectively. FXIII activation is delayed relative to FPA release. Around clot time in hemophilia whole blood, FPB levels are only ~1μ M and FPA levels are 8 fold higher. In normal whole blood at clot time, FPA release is only 2.5 fold higher than FPB release, potentially suggesting that there is an essential correlation of time dependence between FPA, FPB and FXIII activation to form a stable clot. Whereas, this pattern of fibrin formation is altered in the hemophilia state. Clots from hemophilia whole blood are more soluble as evidenced by a 43% decrease in fibrin mass compared to clots from healthy individuals. Overall, data indicate that hemophilia clots are weaker due to decreased lateral association (FPB release) and are mostly composed of fibrin units that are only partially cross-linked. Thus, suggesting that FXIII activation and FPB release play crucial roles in the pathology of hemophilia and the fragile nature of blood clots in hemophiliacs.

The Antithrombogenecity of Silk Fibroin Films Blending with Citric Acid and PVA

The citric acid (CA) was added to the silk fibroin and Polyvinyl Alcohol (PVA) copolymer system. The blending 4% PVA was for improving the mechanical properties of the silk fibroin films. Citric acid amount varies from 0.5% to 2% of weight. The antithrombogenecity was characterized by in vitro clot time of prothrombin time (PT), the Activated partial thromboplastin time (APTT) and thrombin time (TT). The mechanical properties of the blending films were measured and the tensile strength and breaking elongation of films reached to 25.3MPa and 102%. The anticoagulant property results indicated that APTT and TT prolonged significantly, but PT has no effective change. We discussed the relationship between the structure and properties and proposed an anticoagulant mechanism.