COVID-19: A case for plasma derived natural anticoagulants?

Convalescent plasma was proposed for passive immunization against COVID-19; but so far there are conflicting results and still open questions. However, besides antibodies, other plasma proteins may be good candidates for further research and application. Thromboinflammation frequently complicates severe COVID-19, and classical anticoagulants like heparins seem to have limited effect. The natural protease inhibitors antithrombin III (ATIII), α1-antitrypsin (α1-AT) and α2-macroglobulin (α2-M), which are found decreased in severe COVD-19, play a crucial role in prothrombotic and inflammatory pathways. While ATIII and α1-AT are licensed as commercially prepared therapeutic concentrates, there is no preparation of α2-M available. The diagnostic, prognostic, and even therapeutic potential of plasma protease inhibitors should be further explored.

Thromboinflammation in COVID-19: Can α2 -macroglobulin help to control the fire?

The complex COVID-19-associated coagulopathy appears to impair prognosis. Recently, we presented the hypothesis that children are to some extent protected by higher α2 -macroglobulin (α2 -M) levels from severe COVID-19. In addition to endothelial cells, thrombin, and platelets, neutrophil granulocytes also appear to play an important role. Neutrophils extrude extracellular nets, which are histone- and protease-coated web-like DNA structures; activate coagulation and platelets; and release radicals and proteases such as elastase. The unique phylogenetically ancient and "versatile" inhibitor α2 -M contributes particularly during childhood to the antithrombin activity of plasma, binds a broad spectrum of proteases, and interacts with other mediators of inflammation such as cytokines. It is suggested that the scope of basic research and clinical studies would include the potential role of α2 -M in COVID-19.

Evaluation of the in vitro Function of Platelet Concentrates from Pooled Buffy Coats or Apheresis.

BACKGROUND: Platelet concentrates play an important role in transfusion medicine. Their short lifespan and lack of robustness require efforts to ensure adequate product quality. In this study, we compared the in vitro quality of the main concentrate types, pooled platelet concentrate (PPC) from whole blood donations, and platelet concentrate from single-donor apheresis (APC). METHODS: Twenty PPCs and 20 APCs prepared in plasma were analyzed on days 2, 4, and 7 of storage. Variables related to metabolism, degranulation, platelet aggregation, P-selectin expression, and annexin V binding were analyzed. Morphology was assessed by transmission electron microscopy of ultrathin sections. A microfluidic device was applied to test the effects of shear stress on platelet function. RESULTS: The metabolic parameters indicated stable storage conditions throughout the 7-day period. The resting discoid form was the prevailing morphology on days 2 and 4 in the PPCs and APCs. Chemokine release and receptor shedding of soluble P-selectin and soluble CD40L equally increased in PPCs and APCs. Aggregation responses to ADP and collagen were heterogeneous, with marked losses in collagen responsiveness on day 4 in individual concentrates. Baseline expression of P-selectin in PPCs and APCs was low, and inducibility of P-selectin was well preserved until day 4. Under shear stress, equal adhesiveness and stability were found with platelets from PPCs and APCs. CONCLUSIONS: Platelets from PPCs and APCs showed similar in vitro function and stability parameters. However, platelet concentrates presented a high variability and individual concentrates an impaired functional capability. Identifying the factors contributing to this would help increase product reliability.

Kreuth V initiative: European consensus proposals for treatment of hemophilia using standard products, extended half-life coagulation factor concentrates and non-replacement therapies.

This report contains the updated consensus recommendations for optimal hemophilia care produced in 2019 by three Working Groups (WG) on behalf of the European Directorate for Quality of Medicines and Healthcare in the frame of the Kreuth V Initiative. WG1 recommended access to prophylaxis for all patients, the achievement of plasma factor trough levels of at least 3-5% when extended half-life factor VIII (FVIII) and FIX products are used, a personalized treatment regimen, and a choice of chromogenic assays for treatment monitoring. It was also emphasized that innovative therapies should be supervised by hemophilia comprehensive care centers. WG2 recommended mandatory collection of postmarketing data to assure the long-term safety and efficacy of new hemophilia therapies, the establishment of national patient registries including the core data recommended by the European Medicines Agency and the International Society on Thrombosis and Haemostasis, with adequate support under public control, and greater collaboration to facilitate a comprehensive data evaluation throughout Europe. WG3 discussed methodological aspects of hemophilia care in the context of access decisions, particularly for innovative therapies, and recommended that clinical studies should be designed to provide the quality of evidence needed by regulatory authorities, HTA bodies and healthcare providers. The dialogue between all stakeholders in hemophilia care and patient organizations should be fostered to implement these recommendations.

Free factor XIII activation peptide (fAP-FXIII) is a regulator of factor XIII activity via factor XIII-B.

In a factor XIIIa (FXIIIa) generation assay with recombinant FXIII-A2 (rFXIII-A2 ) free FXIII activation peptide (fAP-FXII) prolonged the time to peak (TTP) but did not affect the area under the curve (AUC) or concentration at peak (CP). Addition of recombinant factorXIII-B2 (rFXIII-B2 ) restored the characteristics of the FXIIIa generation parameters (AUC, TTP and CP) to those observed for plasma FXIII (FXIII-A2 B2 ). FXIII-A2 B2 reconstituted from rFXIII-A2 and rFXIII-B2 showed a similar effect on AUC, TTP and CP in the presence of fAP-FXII as observed for plasma FXIII-A2 B2 , indicating a role for FXIII-B in this observation. An effect of fAP-FXIII on thrombin, the proteolytic activator of FXIII, was excluded by thrombin generation assays and clotting experiments. In a purified system, fAP-FXIII did not interfere with the FXIIIa activity development of thrombin-cleaved rFXIII-A2 (rFXIII-A2 ') also excluding direct inhibition of FXIIIa. However, FXIIIa activity development of FXIII-A2 'B2 was inhibited in a concentration-dependent manner by fAP-FXIII, indicating that an interaction between AP-FXIII and FXIII-B2 contributes to the overall stability of FXIII-A2 'B2 . In addition to its well-known role, FXIII-B also contributes to FXIII-A2 B2 stability or dissociation depending on fAP-FXIII and calcium concentrations.

[Authorization of tissue and blood stem cell preparations for hematopoietic reconstitution: Documentation of clinical and nonclinical data in a central expert report]. / Genehmigungsverfahren für Gewebezubereitungen und Blutstammzellzubereitungen zur hämatopoetischen Rekonstitution: Dokumentation klinischer und nicht klinischer Daten mittels eines Zentralgutachtens.

In order to address the European Directive 2004/23 on human tissues and cells, the authorization obligation for tissue and blood stem cell preparations was introduced (§ 21a AMG) in the year 2007 in the German medicinal products act. Stem cell transplantation for hematopoietic reconstitution has been in use for decades and is well established for the treatment of many malignancies. The manufacture of stem cell preparations varies, but in terms of hematopoietic reconstitution, different products are intended for the same indication. Taking these aspects into account, it was considered inappropriate that every single applicant should provide their own documentation, including an expert report on clinical and nonclinical data. Consequently, the idea came up to create a central expert report, to which all applicants could refer and would include relevant clinical and nonclinical data according to current knowledge. A central expert report was therefore generated, called the "Gemeinsame Stellungnahme der Fachgesellschaften Deutsche Gesellschaft für Transfusionsmedizin und Immunhämatologie (DGTI), Deutsche Gesellschaft für Hämatologie und Onkologie (DGH) und Gesellschaft für Pädiatrische Onkologie und Hämatologie (GPOH)". Applicants are allowed to refer to this central expert report provided their stem cell product is comparable with the cell preparations included in the report. In order to represent current knowledge, the content of the central expert report was already reworked once, but should be updated regularly.

Significance of platelet and monocyte activation for therapeutic immunoglobulin-induced thromboembolism.

OBJECTIVES: Thromboembolic events (TEE) in patients receiving infusions of intravenous immunoglobulin (IVIG) products have recently been associated with contaminating factor XIa. We studied whether platelet and monocyte activation could also be involved. METHODS: Twenty IVIG samples from five manufacturers were tested for the induction of visible whole blood clot formation. A selection of TEE-associated and not associated lots was further analyzed for effects on thromboelastometry, platelet activation and adhesion, as well as monocyte tissue factor surface expression. Pure factor XIa was included for comparison. Western blotting was applied to analyze anti-CD154-reactive proteins in IVIG. RESULTS: In whole blood, IVIG enhanced macroscopic clotting additively with factor XIa. In monocytes, all IVIG products induced the FcγRII-dependent tissue factor expression to a similar extent, which was not affected by addition of factor XIa. Testing platelet aggregation, IVIG strengthened the ADP and TRAP-6-elicited response. Furthermore, IVIG increased platelet-monocyte adhesion and annexin V binding to platelet microvesicles, and promoted platelet adhesion to IVIG-coated surfaces. The strongest effects were observed with TEE-associated lots. CD154-related proteins were detected in all IVIG products. CD154-related high molecular weight complexes were particularly found in the TEE-associated IVIG. In platelet aggregation, recombinant soluble CD154 enhanced aggregate formation and stability. CONCLUSION: Our data demonstrate that IVIG modulate platelet and monocyte activation and can thereby affect the hemostatic balance. These effects are either additive to or independent from factor XIa. CD154-related proteins are assumed to be involved in these interactions, the mechanism of which needs to be elucidated in further studies.

Factor XIIIa generation assay: a tool for studying factor XIII function in plasma.

Triggering the extrinsic coagulation pathway in plasma and using a fluorogenic factor XIIIa (FXIIIa) substrate for continuously monitoring FXIIIa activity, an FXIIIa generation curve is obtained. The parameters area under the curve (AUC), time to peak (TTP), and concentration at peak (CP) were calculated. In dilutions of normal plasma in FXIII-deficient plasma, AUC and CP showed linear dose-response relationships, whereas TTP increased from 9.9 min for 25% FXIII to 11.6 min for 100% FXIII. Three FXIII-A preparations (rFXIII, rFXIII(V34L), and cellular FXIII [cFXIII]) showed a linear dose response for AUC and CP. The TTP increased slightly for rFXIII from 13.5 to 15.0 min, but surprisingly for cFXIII TTP increased concentration dependently from 13.5 to 28.7 min. Adding 5 µg/ml FXIII-B at a concentration of 1U of FXIII-A increased the AUC for rFXIII(V34L) and cFXIII by approximately 20% and accelerated TTP from 27.3 to 20.8 min for cFVIII, indicating a supportive function of FXIII-B in orientating cFXIII-A for thrombin cleavage. A commercial assay quantifying FXIII after complete activation in a restricted time window did not reveal differences in the cFXIII preparation with or without FXIII-B. The FXIIIa generation assay provides additional information about activation and function of FXIII. This advantage was underlined in experiments with an irreversible FXIIIa inhibitor.

Inducible expression of tissue factor in small-cell lung cancer: impact on morphology and matrix metalloproteinase secretion.

PURPOSE: Tissue factor (TF), the transmembrane receptor for factor VIIa (FVIIa), has key regulatory functions in coagulation as well as in tumour progression and metastasis. Small-cell lung cancer (SCLC) metastasises more aggressively than non-small-cell lung cancer (NSCLC). Previously, we described the transition of SCLC cell line H69 to adherent growth and TF expression. Here, we explored the differential expression of TF and its functional impact on morphology and matrix metalloproteinase (MMP) secretion. METHODS: The constitutional TF expression was evaluated in a panel of established NSCLC and SCLC cell lines. Furthermore, in three stress-selected adherent SCLC H69 cells, TF and MMP expressions were determined by mRNA, protein, and activity measurements. RNA interference-mediated TF down-regulation and FVIIa stimulation were used to study the impact of TF on cellular functions. RESULTS: NSCLC cells expressed high TF antigen (median 3.75 ng/mg; range 0.31-65.2 ng/mg protein, n = 8), while SCLC expressed none or low TF (median 0.07 ng/mg; range 0-0.39 ng/mg protein, n = 6). However, selected H69 adherent cells markedly expressed TF (range: 4.8-44.3 ng/mg protein, n = 3) and secreted MMP-2 and MMP-9. FVIIa stimulated MMP-2 and MMP-9 secretion in H69adh cells, whereas TF down-regulation diminished MMP-2 and MMP-9 expression and promoted reversion to suspension growth. CONCLUSIONS: Our data show the significance of TF expression in the reversible growth phenotype of H69. Because TF, MMP expression, and adherence are highly relevant to cancer metastasis, this study suggests a novel mechanism of adaptation, thereby adding to the understanding of SCLC biology and its aggressiveness.