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Heparin-induced thrombocytopenia (HIT) is the most life-threatening adverse effect of heparin therapy and is provoked by the development of drug-dependent antibodies. It occurs more frequently in patients with cardiac or orthopedic surgery or severe circulatory diseases, and the risk depends on the patient pathological status. As heparin is an anticoagulant used for treating thrombotic events or their risk, this iatrogenic complication has a paradoxal effect as it can induce thromboembolic diseases, frequently associated to severe morbidity or fatal outcomes. Diagnosis involves clinical evaluation of disease probability and laboratory tools for testing the presence of heparin-dependent antibodies with immunoassays or their capability to activate platelets with functional assays. Antibodies developed when stoichiometric complexes of platelet factor 4 (PF4) with heparin are formed during therapy. In few cases non-platelet factor 4 antigens can be involved. Antibodies can remain asymptomatic, but pathogenicity occurs in the presence of high concentrations of IgG isotype antibodies, with high avidity: they target and activate platelets or endothelial cells exposing heparin-PF4 (HPF4) complexes and produce thrombocytopenia and sometimes thrombosis. Risk factors which favor the development of antibodies and their pathological effect are discussed. The present understanding of mechanisms underlying disease development and diagnostic strategies of this heparin adverse effect is presented.

Coagulation Factor V (FV) is a key factor for regulating blood coagulation cascade, and it acts at the crossroads of the intrinsic and extrinsic pathways. It shows a dual activity as the procoagulant cofactor for Factor Xa in the prothrombinase complex, but it also supports an anticoagulant activity in combination with TFPI and Protein S. Its rapid cleavage by Activated Protein C (APC) complexed with Free Protein S (FPS), in presence of phospholipids and calcium, inhibits its activity and limits the propagation of blood coagulation, keeping it to where it is beneficial. Rapid inactivation of active FV by APC-FPS is essential for preventing the risk of thrombosis development. In 1993, Dahlbäck and coworkers reported an inherited disorder characterized by activated protein C resistance (APC-R) and associated to an increased occurrence of thromboembolic events in affected families. In 1994 Bertina demonstrated that this diathesis resulted from a Factor V mutation (R506Q), rendering this factor resistant to inactivation by APC. This mutated Factor V was called Factor V Leiden (FV-L). APTT based assays and molecular biology methods for detecting the mutation were developed, but these methods are only qualitative and classify tested individuals as normals, heterozygous or homozygous for the coagulation defect. Our group developed a quantitative assay for FV-L, which is described in this report, along with its performances. This assay allows to quantitate specifically FV-L coagulant activity, and to graduate its amount in heterozygous or homozygous patients. FV-L is absent in normal individuals and present in homozygous or heterozygous patients, accounting respectively for 100 % or 50 % of blood FV. Its amount is compared with FV clotting activity or antigenic concentration. Measured FV-L activities overlap between heterozygous patients with high FV and homozygous ones with low FV levels. This assay allows to better discriminate for the FV-L associated thrombotic risk, which depends on the effective FV-L concentration rather than on patients’ genetic status. This expectation is supported by literature review, which shows that FV-L concentrations correlate with presence of platelet released microparticles in patients carrying that mutation.