Altered fibrin clot structure contributes to thrombosis risk in severe COVID-19

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. Here, we demonstrate altered levels of factor XII (FXII) and its activation products in two independent cohorts of critically ill COVID-19 patients in comparison to patients suffering from severe acute respiratory distress syndrome due to influenza virus (ARDS-influenza). Compatible with this data, we report rapid consumption of FXII in COVID-19, but not in ARDS-influenza, plasma. Interestingly, the kaolin clotting time was not prolonged in COVID-19 as compared to ARDS-influenza. Using confocal and electron microscopy, we show that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggers formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, we observed clot lysis in 30% of COVID-19 patients and 84% of ARDS influenza subjects. Analysis of lung tissue sections revealed wide-spread extra- and intra-vascular compact fibrin deposits in COVID-19. Together, our results indicate that elevated fibrinogen levels and increased FXII activation rate promote thrombosis and thrombolysis resistance via enhanced thrombus formation and stability in COVID-19.

Antiphospholipid Antibodies in Iraqi Women with Recurrent Mid-Trimester Abortions

ABSTRACT Purpose: Antiphospholipid antibodies are often associated with recurrent pregnancy loss, and although many studies have addressed this association in Western countries, such studies are not so frequent from developing countries. The current study aims to determine the frequency of Antiphospholipid antibodies (Anticardiolipin antibodies and Lupus anticoagulant) among Iraqi women with recurrent mid-trimester abortions and to evaluate various tests used for their detection. Materials and Methods: Two hundred women with recurrent mid-trimester abortions were randomly enrolled from a main referral center in Baghdad-Iraq. The enrollees had their IgG and IgM anticardiolipin antibodies assayed by ELISA, and Lupus anticoagulant by a combination of the following screening tests: Activated Partial Thromboplastine Time (APTT), and Partial Thromboplastine Time-LA (PTT-LA), Kaolin Clotting Time (KCT) and confirmation was made by Hexagonal phospholipid neutralization test. Results: The women were aged between 19 and 45 years (median 30 years). Fifty three (26.5%) had one or both anticardiolipin antibodies present, while 27 (13.5%) were positive for lupus anticoagulant. The KCT and KCT index appeared to be the most sensitive tests, while the KCT index and APTT were the most specific for Lupus anticoagulant. Patients with antiphospholipid antibodies had higher rates of history of thrombosis, thrombocytopenia and family history of recurrent abortion (P = 0.0009, 0.0056 and 0.0003 respectively). Conclusions: Antiphospholipid antibodies constitute an important cause of recurrent mid-trimester abortion in Iraqi women, with frequencies intermediate between Western and Indian reports. While thrombocytopenia and thrombosis are well documented associations of antiphospholipid antibodies, the significant association with family history of recurrent fetal loss is intriguing and requires further scrutiny.

Lupus Anticoagulants and Thrombosis: Clinical Association of Different Coagulation and Immunologic Tests

SummaryThe dilute Russell’s viper venom time (dRVVT) and the kaolin clotting time (KCT) are two among the most commonly used coagulation tests for the detection of lupus anticoagulants. The dRVVT seems superior to the KCT in identifying LA-positive patients at risk of thrombosis. However, this relationship is greatly influenced by both the source of reagents and the instrumentation employed to carry out the assays. Therefore, 4 dRVVTs (“home-made” dRVVT, DVV test, Bioclot LA, LA Screen), and one KCT (Kaoclot) were performed in two centers and compared for their retrospective correlation with the thrombotic complications of 72 patients with a previously established diagnosis of lupus anticoagulants. Two other assays (“home-made” KCT, and Colloidal Silica Clotting Time, CSCT) were performed in one of the two centers, and compared with Kaoclot for their clinical correlations in the same population of patients, 44 of whom (61%) had suffered from arterial and/or venous thrombosis. A rather good degree of inter-laboratory and inter-assay correlations of the different tests was found. However, a statistically significant association with thrombosis was found only with the coagulation profile generated using the “homemade” dRVVT. When the commercially available dRVVTs were used, none of the coagulation profiles remained associated with thrombosis. When the assays were analyzed separately, the association with thrombosis was statistically significant for LA screen (p = 0.0019), DVV test (p = 0.0043), and Bioclot (p = 0.0255), and of borderline significance for the “home-made” dRVVT (p = 0.0503) in one center. This last assay was also significantly associated with thrombosis in the other center (p = 0.0139). When venous and arterial thrombosis were considered separately, DVV test was statistically associated with venous thrombosis in both centers (p = 0.0076 and p = 0.0187, respectively), and LA screen in one center (p = 0.0303). No dRVVT was found to correlate with arterial thrombosis. Kaoclot, Colloidal Silica Clotting Time, and the “home-made” KCT did not correlate with thrombosis. The prevalence of IgG and/or IgM antibodies to cardiolipin, β2-glycoprotein I and prothrombin were 74%, 86% and 85%, respectively. Increased titers of IgG anticardiolipin antibodies were associated with arterial thrombosis (p = 0.0375), whereas IgM anti-β2-glycoprotein I antibodies were associated with venous thrombosis (p = 0.0433). In conclusion, these retrospective data support the notion that the dRVVT, rather than other coagulation or ELISA tests, are able to identify lupus anticoagulant-positive patients at risk of thrombosis. This property appears common to several commercially available dRVVT kits, making this type of assay the ideal target of future efforts of laboratory standardization.

dRVVT Is more Sensitive than KCT or TTI for Detecting Lupus Anticoagulant Activity of Anti-β2-glycoprotein I Autoantibodies

SummaryAnti-β2-glycoprotein I (β2-GPI) antibodies behave as classical Lupus Anticoagulants (LA), as they inhibit phospholipid-dependent coagulation reactions and their activity disappears in the presence of excess exogenous phospholipids (PLs). We have recently shown that a certain amount of PLs in the dilute Russell Viper Venom Time (dRVVT) test system is required to express LA activity of anti β2-GPI antibodies. We have now extended this observation to two other tests, i.e., Kaolin Clotting Time (KCT) in which PLs are not added, and Tissue Thromboplastin Inhibition test (TTI) in which PLs are extremely diluted. In fact, affinity-purified antibody preparations from 5 patients with antiphospholipid syndrome did not express or only weakly expressed anticoagulant activity in both tests; the mean ratios of coagulation times obtained with purified antibodies and that of control buffer were 1.11 and 1.0 for KCT and TTI, respectively. On the contrary, the mean ratios in dRVVT were 1.31 and 1.49 at a PLs dilution of 1:8 and 1:64, respectively. Therefore, the presence of LA activity due to autoantibodies to β2-GPI is characterized by a positive dRVVT and negative or only weakly positive KCT and TTI.

Prothrombin as cofactor for antiphospholipids

Prothrombin is a common antigenic target of antiphospholipid antibodies, since anti-prothrombin antibodies are detected in about 50-90% of the patients. To allow proper immune recognition, prothrombin must be adsorbed on suitable anionic surfaces. The epitope(s) have not yet been identified: the majority of anti-prothrombin antibodies appear to be of poly- or oligoclonal nature. Anti-prothrombin antibodies, either alone or in combination with anti-β2-glycoprotein I antibodies, are responsible for the lupus anticoagulant activity of about 75% of the cases of phospholipid-dependent inhibitors of coagulation. The two antibodies may be discriminated by means of specific coagulation profiles generated by the comparison of the ratio of the Kaolin Clotting Time (KCT) and the dilute Russell's Viper Venom Time (dRVVT): the KCT profile, which mainly reflects the presence of anti-prothrombin antibodies and the dRVVT profile, which is mostly associated with anti-β2-glycoprotein I antibodies. This distinction, although somewhat artificial, may be clinically useful, since the KCT profile identifies patients at low risk to develop thrombosis. Similarly, most of the studies that measured anti-prothrombin antibodies by ELISA failed to find a significant association with thrombosis. In conclusion, the clinical relevance of these antibodies has not yet been established.