Protection of β2GPI Deficient Mice from Thrombosis May Reflect a Platelet Activation Defect

Introduction Antiphospholipid syndrome (APS) is an autoimmune disorder caused by "antiphospholipid" antibodies (aPL) directed against β2-glycoprotein I (β2GPI). Although β2GPI is proposed to have both anti- and procoagulant properties in vitro, its physiological role in coagulation in vivo is not well understood. Previous studies have shown that β2GPI deficient mice display impaired thrombin generation but failed to demonstrate an anticoagulant role in vivo. Recent findings from our laboratory demonstrated that β2GPI-deficient mice (APOH -/-) developed by CRISPR/Cas9 had shown delayed time to thrombosis as evidenced by prolonged clotting times in carotid artery occlusion models. However, no mechanism was identified for the delayed time to thrombosis phenotype. Methods Venous thrombosis by complete occlusion of the IVC was performed as described previously (Wrobleski et al., 2011). In brief, an abdominal incision was made on anesthetized mice to visualize IVC. The activated partial thromboplastin time (aPTT); prothrombin time (PT) and Tissue factor-induced thrombin generation time (TGT) were performed as described previously (Stavrou et al., 2014). Protein C activity was performed according to manufacturer's recommendations (Chromogenix Coamatic® Protein C, Diapharma). To assess platelet activation, platelets were isolated under resting conditions using retro-orbital blood and the final washed platelet pellet was resuspended in Tyrodes solution. Platelets were stimulated with 0.25 U/ml thrombin and incubated with 1 µL of CD62P-FITC or Oregon Green conjugated-fibrinogen for 30 minutes. Platelets were fixed with 100 μL 2% formalin, and quantification of platelet surface P-selectin and fibrinogen binding to activated platelet GPIIb/IIIa was performed by flow cytometry (Accuri Flow Cytometer, BD Biosciences). For mouse tail vein bleeding time assays, mice were anesthetized and the tail transected approximately 5 mm from the tip. The tail was then placed in a 50 ml falcon tube containing saline pre-warmed to 37°C. The time to cessation of bleeding was determined visually. All experiments were performed on 8-12 week old mice. Results aPTT and PT results for APOH +/+ and APOH -/- mice were 41.82 ± 1.174 and 41.38 ± 2.026 seconds, and 14.55 ± 2.262 and 11.30 ± 0.578 seconds, respectively (NS). After IVC ligation, thrombi in APOH -/- mice had a mean weight of 16.94 ± 1.782 mg compared to 27.69 ± 1.725 mg in APOH +/+ mice littermates (P = 0.0002, Figure A). Thrombin generation induced by either tissue factor or aPPT reagent showed peak thrombin generation at 15 min with no difference between APOH +/+ and APOH -/- mice. Likewise, no significant differences were observed in percent Protein C activity levels between APOH +/+ (8.058 ± 1.433) and APOH -/- (6.453 ± 0.924). Stimulation with 0.25 U/ml thrombin resulted in significantly greater expression of P-selectin) on platelets of APOH +/+ (161.3 ± 30.62 MFI) compared to those from APOH -/- mice (54.47 ± 9.721 MFI) (P= 0.0101;Figure 1B). Likewise, there was significantly greater fibrinogen binding to stimulated platelets from APOH +/+ (129.7 ± 32.21 MFI) compared to APOH-/- mice (35.30 ± 2.144 MFI) with P = 0.0111 (Figure 1C). Finally, consistent with platelet activation studies, tail vein bleeding times were mildly, but significantly prolonged in APOH -/- mice (192.3 ± 45.86 sec) compared to APOH+/+ mice (95.14 ± 9.582 sec) with P = 0.. Conclusion The effects of β2GPI, if any, on coagulation processes is controversial, and has not been thoroughly studied in β2GPI deficient animals. The results presented here suggest that the smaller thrombi that form in mice deficient in β2GPI following IVC occlusion may reflect a mild platelet function defect. This hypothesis is supported by diminished P-selectin expression and fibrinogen binding in response to 0.25 U/ml thrombin by platelets from β2GPI deficient mice compared to wild-type littermates. Moreover, tail vein bleeding times were mildly prolonged in β2GPI deficient mice. Taken together, these studies suggest that β2GPI may actually contribute to hemostasis by supporting platelet responses to low concentrations of thrombin. Additional studies are needed to characterize these effects in detail. Figure 1 Figure 1. Disclosures McCrae: Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria; Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy.

Case Report: Successful Long-Term Management of a Low-Birth Weight Preterm Infant With Compound Heterozygous Protein C Deficiency With Subcutaneous Protein C Concentrate Up to Adolescence

Homozygous/compound heterozygous forms of congenital protein C deficiency are often associated with severe antenatal and postnatal thrombotic or hemorrhagic complications. Protein C deficiency frequently leads to severe adverse outcomes like blindness and neurodevelopmental delay in children and may even lead to death. The most widely used long-term postnatal treatment consists of oral anticoagulation with vitamin K antagonists (e.g., warfarin), which is supplemented with protein C concentrate in acute phases. Subcutaneous infusions have been described in infants mostly from 2 months of age after severe postnatal thrombosis, but not in newborns or premature infants without thromboembolism. We report the first case of a compound heterozygous protein C-deficient preterm infant, born at 31+5 weeks of gestation to parents with heterozygous protein C deficiency (protein C activity 0.9% at birth). We focus on both prenatal and perinatal management including antithrombotic treatment during pregnancy, the cesarean section, and continuous postnatal intravenous and consecutive subcutaneous therapy with protein C concentrate followed by a change of therapy to direct oral anticoagulants (DOACs) (apixaban). We report successful home treatment with subcutaneous protein C concentrate substitution overnight (target protein C activity >25%) without complication up to 12.5 years of age. We propose that early planned cesarean section at 32 or preferably 34 weeks of gestation limits potential maternal side effects of anticoagulation with vitamin K antagonists and reduces fetal thromboembolic complications during late pregnancy. Intravenously administered protein C and early switch to subcutaneous infusions (reaching about 3 kg body weight) resulted in sufficient protein C activity and has guaranteed an excellent quality of life without any history of thrombosis for 13 years now. In older children with protein C deficiency, as in our case, DOACs could be a new therapeutic option.

NECROSE CUTANEE REVELANT UN DEFICIT CONGENITAL EN PROTEINE C : A PROPOS DE 3 CAS

Neonatal skin necrosis in the context of a congenital homozygous protein C deficiency is a rare inherited autosomal recessive disorder, it is characterized by rapidly extensive necrotic patches occurring a few hours after birth in a newborn who doesnt present any hemodynamic disorder. The diagnosis is based on the assay of protein C activity which is collapsed or even undetectable. Early diagnosis and replacement therapy are the mainstays of management before the onset of disseminated intravascular coagulation. We report three cases of newborns presenting with DIC in the context of protein C deficiency and the course of which was fatal.

Effect of Pulmonary Tuberculosis on Protein C, S, and Antithrombin-III among Therapy-naïve Ghanaian Adults; A Comparative Cross-Sectional Study

Background. Tuberculosis (TB) constitutes a global emergency as it affects one-third of the world’s inhabitants. Although pulmonary Tuberculosis (PTB) is curable, immunological responses to the infection induce several haematological derangements. This study evaluated the effect of PTB on Protein C, Protein S, Antithrombin-III, and blood count parameters. Methods. Ninety adults with ages ≥18 years were purposively recruited: 60 PTB patients and 30 non-TB controls. All patients were diagnosed with sputum GeneXpert MTB/Rif assay. Blood specimens were collected from each participant for Protein C, S, Antithrombin-III and complete blood count. Results. Pulmonary TB was associated with significantly reduced Protein C activity (101.46 [87.61-128.3] vs 121.44 [99.50-149.8] IU/L, p=0.038), RBC (3.88±0.91 vs 4.80±0.55, p<0.0001), HgB (10.24±2.42 vs 11.78±1.42, p=0.0019), HCT (32.21±7.79 vs 42.05±4.97, p<0.0001), MCV (83.80 [79.33-90.08] vs 89.00 [83.75-92.00], p=0.0133) and PDW (12.95 [10.73-15.00] vs 15.30 [14.18-15.93], p<0.0001) compared to controls. Conversely, PTB patients were associated with significantly increased MCH (26.83±4.33 vs 24.59±1.99, p=0.0086), TWBC (7.76 [6.06-9.78] vs 6.50 [4.85-7.50], p=0.0047), Abs. GRAN (5.27 [3.30-6.71] vs 3.75 [2.48-4.75], p=0.0226), RDW-CV (13.70 [13.20-15.43] vs 12.95 [12.50-13.65], p<0.0001), MCHC (32.10 [28.70-35.63] vs 27.85 [27.40-28.53], p<0.0001) and MPV (8.3 [6.7-9.7] vs 7.0 [6.4-7.5], p=0.0027) compared to controls. The PTB patients were disproportionately affected with anaemia (91.7%, p=0.001), erythrocytopenia (75.0%; p≤0.001) and reduced HCT (80.0%, p≤0.001). The frequency of thrombocytosis, leucocytosis, and granulocytosis (50.0%, p=0.013; 23.3%, p=0.013; 18.3%, p=0.025; respectively) in PTB patients were significantly higher than in controls. Conclusion. Our findings suggest that PTB predisposes patients to hypercoagulability, with a significant reduction in Protein C activity but not Protein S and antithrombin-III. The condition causes derangements in erythrocytes, leucocytes, and thrombocytes, and disproportionately causes anaemia. Protein C activity and complete blood count are useful in the management of PTB and should be included in the routine workup for patients.

Plasmatic and cell components of hemostasis in patients with serious burned wounds: studying at early period of disease

We studied coagulation parameters of severely burned patients at early period of disease (1–10 days). All patients had II–III degree burned wound, varied from 22 to 75 % total body surface area (median 40 % [35; 60]). Patients were divided into two groups: survival (35 patients, 1th group) and lethal (19 patients, 2nd group) outcomes. During burned shock (1st‑2nd day) 1th group had normal activated partial thromboplastin time, fibrinogen concentration, protein C activity, whereas in 2nd group these parameters were reliably abnormal. In both groups we noticed significant decay of antitrombin III activity and increase of D-dimer, followed by very low integrity of platelets. We found correlation between morphofunctional platelet rate and blood clotting activity. At 3rd day all patients showed significant increase of fibrinogen concentration without change of other parameters. At 10th day patients with survival outcome normalized antitrombin III activity and had particular recover of platelet integrity, patients with lethal outcomes did not have such effects. Values of D-dimer, antitrombin III and protein C activity reliably differed between the groups throughout the observation period. The decrease of antitrombin III and protein C activity in the first day after the burn is critical. Reducing the activity of antitrombin III less than 75 % is a predictor of adverse outcome.

PROC Promoter Single Nucleotide Polymorphisms Associated With Low Protein C Activity But Not Increased Risk of Thromboembolism in Pediatric Population

Protein C (PC) deficiency, caused by mutations of the PROC gene, is a common inherited risk factor of thromboembolism (TE) among Thai people. This study aimed to investigate the association of 3 single nucleotide polymorphisms (SNPs; −1654 C/T, −1641 A/G, −1461A/T) at the PROC promoter region with PC activity and the risk of developing TE. A total of 216 patient s with TE, diagnosed at aged 0 to 20 years, and 102 healthy adults were enrolled. The SNPs were identified by Sanger sequencing. Protein C activity was measured using an automated functional clotting assay. Linear and logistic regression analyses were used to determine the association of SNPs with PC activity and the risk of TE. Patients and controls with homozygous TAA (119.6% ± 26.1%) and CGT haplotypes (102.7% ± 22.6%) had significantly lower PC activity than those with a homozygous CAA haplotype (140.4% ± 44.9%); P = .027 and .016, respectively. However, none of these haplotypes increased the risk of TE. This study suggested that the 3 PROC promoter SNPs were shown to be associated with lower PC activity but did not increase the risk of TE.