Hemostatic Abnormalities

Hemostatic Abnormalities

INTRODUCTION

Hemostasis is a process which occurs to prevent and stop bleeding, mainly to keep blood within a damaged blood vessel. This involves coagulation, blood changing from a liquid to a gel. The endothelial cells of intact vessels prevent blood clotting with a heparin-like molecule and thrombomodulin and prevent platelet aggregation with nitric oxide and prostacyclin.

When endothelial injury occurs, the endothelial cells stop secretion of coagulation and aggregation inhibitors and instead secrete von Willebrand factor, which initiate the maintenance of hemostasis after injury.

Hemostasis has three major steps: 1) vascular spasm 2) temporary formation of platelet plug and 3) blood coagulation or formation of a fibrin clot. These processes seal the hole until tissues are recovered.

Haem=Blood        Stasis=stop

Hemostasis or Haemostasis, both of them is the same.

Definition: Hemostasis is a process occurs to prevent or stop bleeding when the blood is present outside the blood vessels and it has 3 steps. First is vascular spasm, second is platelet plug formation and third is clot formation.

Hemostasis Pathways

Hemostasis process reflects a balance between procoagulant and anticoagulant factors, working in concert to maintain blood flow within the vascular components or promoting the formation of blood clots following vascular injury. For convenience, this process is often divided into three phases: primary hemostasis, secondary hemostasis, and fibrinolysis.

The process of hemostasis also enables repair after vascular injury, promotes vessel healing, and maintains vessel integrity. Disturbances in procoagulant or anticoagulant factors may delay clot formation, vessel repair, and result in a bleeding disorder or increased blood clotting producing thrombosis.

Components of Normal Hemostasis

  • Primary Hemostasis

Primary hemostasis begins after endothelial damage, comprises the process of adhesion of platelet, activation and aggregation to form the platelet plug at the injury site.

Both endothelial cells (EC)—lining the blood vessels—and circulating platelets provide a source of von Willebrand factor (VWF), which is one of the most important components of primary hemostasis.

The main mechanism for regulating plasma VWF size is by proteolysis of the ultra large von Willebrand factor (ULVWF) molecules by ADAMTS13.

This process generates the multimeric forms of VWF present in plasma. VWF has two main functions in hemostasis. First, it mediates the binding of platelets to sub-endothelium and, second, it protects circulating factor VIII:Coagulant (FVIII:C) by binding via the D2 and D3 domains and protecting the FVIII molecule from proteolytic degradation.

After vascular injury occurrence, the EC switch from anticoagulant to procoagulant, so promoting platelet aggregation by releasing the contents of their Weibel-Palade bodies. This leads to enhanced local concentrations of VWF and tissue factor (TF). The released VWF binds to the collagen on the exposed sub-endothelial surface, and is then utilized for platelet binding via the glycoprotein Ib (GPIb) complex. Then the platelet plug is formed.

  • Secondary Hemostasis

Secondary hemostasis involves the activation of the coagulation system and coagulation factors to produce fibrinogen (fibrin) to stabilize the platelet plug.

  • Fibrinolysis

Fibrinolysis is the process whereby the clot is broken down to permit tissue repair. This process requires a number of proteins that facilitate the process. Poor control of fibrinolysis can result in excessive bleeding or accelerate thrombosis development.

Unfortunately, there are few good tests to evaluate the role of fibrinolysis in hemostatic/thrombotic disease. The end products of clot dissolution are represented in plasma by fibrin degradation products such as D-dimer.

More recently, the cell-based model of hemostasis reflecting that blood needs to be exposed to cells containing TF for the clotting process to be initiated.

This model has 3 phases of hemostasis (initiation, amplification, and propagation). The three phases of coagulation occur on different cell surfaces. More specifically, initiation occurs on the TF-bearing cell; amplification on the platelet and propagation on the activated platelet surface.

 

  1. Initiation: TF is the primary physiological initiator of coagulation.

There is data suggesting that TF-bearing cells such as fibroblasts bind to platelet surfaces in an evolving thrombus. A key step in effective initiation of the hemostatic mechanism is bringing FVIIa/TF activity into close proximity to activated platelet surfaces.

Plasma FVII comes into contact with TF-bearing extravascular cells during vascular injury, and is rapidly activated through the extrinsic pathway. To regulate this activation, the FVIIa/TF complex is inhibited by TF Pathway Inhibitor (TFPI) or AT.

  1. Amplification: Once the cell surface has been damaged, the formation of a thrombus involves both platelets and coagulation mechanisms. Platelets adhere at the site of endothelial injury and activated by thrombin, causing them to release alpha granules containing FV and VWF, binding to plasma proteins including VWF, promoting the assembly of procoagulant complexes, and ensuring rapid thrombin generation.
  2. Propagation: During the propagation phase, the “tenase” (FVIIa and FIXa/FVIIIa) and “prothrombinase” (FXa/FVa) complexes are assembled on the platelet surface, thus allowing large-scale thrombin generation to take place. This burst of thrombin is necessary to form a hemostatic fibrin clot.Laboratory Tests
    • Prothrombin Time Test (PT)

    The most commonly performed test of hemostasis and monitors coagulation factors. It is used primarily to establish the presence or absence of particular factor deficiencies, to monitor VKA therapy (as part of the International Normalized Ratio (INR) calculation), and helps in evaluating liver disease.

    It is based on thromboplastin and calcium chloride, which are added to sodium citrate-anticoagulated plasma at 37 °C to produce a clot. This end-point can be determined manually, or more commonly by automated instruments. It is sensitive to changes in vitamin K-dependent factors (FII (Prothrombin), FVII, and FX).

    • International Normalized Ratio (INR)

    The INR is a calculation (INR = (patient PT/MNPT)ISI) generated from the PT and mean normal prothrombin time (MNPT).

    It also requires an international sensitivity index (ISI), which may be supplied by the manufacturer for specific instrument/reagent combinations, or can else be established by the local laboratory for specific instrument, reagent combinations. The laboratory usually establishes the MNPT by calculating the geometric mean of at least 20 individual normal plasma PT values. Despite international standardization, considerable concern remains regarding ongoing high levels of inter-laboratory variation, as generated by different laboratories using the same homogeneous plasma samples.

    • Activated Partial Thromboplastin Time (APTT)

    The activated partial thromboplastin time (APTT) is sensitive to deficiencies of factors of the “intrinsic and common pathways”, namely FII, FV, FVIII, FIX, FX, FXI, FXII, and Fibrinogen.The APTT can be used for inherited or acquired bleeding disorders, which are due to deficiency or defects in these clotting factors (e.g., hemophilia A and B, due respectively to deficiency/defects in FVIII and FIX). The APTT is also used in the hospital setting for monitoring unfractionated heparin (UFH), due to the linear relationship between the APTT and the dosage of UFH throughout the therapeutic range.

    The APTT is also used as a screening test for LA as recommended in the Scientific and Standardization Committee (SCC) of the International Society on Thrombosis and Haemostasis (ISTH) guidelines.

    Here, the APTT is incorporated into a panel of tests either using neat patient plasma or patient plasma mixed with normal plasma, and usually performed with LA-sensitive reagents to maximize the effect of antiphospholipid antibodies (aPL) in prolongation of the APTT, which can then be compared to an APTT performed with a LA-insensitive reagent.

    • Thrombin Time (TT)

    The thrombin time (TT) is a simple and rapid test, which can be used for evaluating presence of UFH in patient plasma due to its high sensitivity. The TT is also sensitive to deficiencies and defects of fibrinogen.

    The breakdown products of fibrinogen/fibrin (Fibrinogen/fibrin degradation products (FDPs), including D-dimer) can also generate prolongation of the TT.

    • Fibrinogen and FXIII

    Fibrinogen can be measured either as a functional (von Clauss) or derived (estimated using the PT).

    As one of the major proteins involved in hemostasis, measurement of fibrinogen is important for detecting congenital abnormalities such as afibrinogenaemia (no fibrinogen) and dysfibrinogenaemia (abnormal fibrinogen), as well as in other conditions such as trauma, disseminated intravascular coagulation (DIC), and liver disease.

    The fibrinogen/fibrin clot is stabilized by factor XIII (FXIII), which cross links the fibrin molecules to help in forming clot.

    The breakdown of this clot (generating FDPs and D-dimer) by the process of fibrinolysis provides a measure of potential thrombosis such as deep vein thrombosis (DVT) or pulmonary embolism (PE).

    Hemostasis Disorders (Thrombophilia)

    Thrombophilia defines disorders of hemostasis, which increase the risk of thrombosis through congenital or acquired deficiencies/defects.

    Thrombosis is a major cause of morbidity and mortality in Western countries.

    1. Congenital Thrombophilia
    • Factor V Leiden (FVL)/Activated Protein C Resistance (APCR)

    FVL is associated with an increased risk of thrombosis, especially DVT, and manifests phenotypically as activated protein C resistance (APCR). APCR is defined as an impaired plasma anticoagulant response to activated protein C (APC), and is essentially an inability to quickly inactivate FVa and/or FVIIIa, which are otherwise formed during coagulation. APCR can be detected by performing a clot-based APCR assay using either APTT or Russell’s viper venom time (RVVT)-based methods, with or without the inclusion of FV-deficient plasma (Table 2). FVL is identified by either APCR or genetic testing specific for FV mutations.

    • Protein C (PC), Protein S (PS), and Antithrombin (AT)

    PC, PS, and AT assays are used to detect congenital deficiencies or acquired defects. Functional and antigenic assays are available, with functional (chromogenic) assays usually performed for PC and AT, and antigenic assays for (free) PS (usually Latex immunoassay (LIA) or enzyme-linked immunosorbent assay (ELISA ); however, clot-based assays are also available for PC and PS).APC, together with its cofactor PS, inactivates the coagulation cofactors FVa and VIIIa, so reducing the thrombin generating capacity of blood. PC, the vitamin K-dependent zymogen of APC, is synthesized in the liver. PC deficiency is usually inherited through an autosomal-dominant manner.

    AT is an important inhibitor of thrombin and other serine proteases such as FXa, FIXa, FXIa, and FXIIa. AT has two important binding sites, one that binds thrombin and the other that binds heparin. When heparin is bound, there is an acceleration of the binding of the serine proteases to the AT molecule. The rapid inactivation of thrombin and anti-Xa in the presence of heparin is referred to as the heparin cofactor activity of AT. Most assays used for the measurement of AT are chromogenic in principle, and these generally represent the best performing of the congenital thrombophilia assays. Test performance for PC and PS tends to be a little less consistent.

    1. Acquired Thrombophilia
    • Antiphospholipid Syndrome (APS)

    APS is a complex disorder associated with thrombosis and pregnancy morbidity. Individuals with APS represent approximately 20% of venous thromboembolism (VTE) cases in the general population, and APS affects an estimated 0.5% of the population. The disorder is characterized by the presence of aPL, notably anticardiolipin antibodies (aCL), anti-β2-glycoprotein I antibodies (a β2GPI).

    • Lupus Anticoagulant (LA)

    The term “lupus anticoagulant” is a misnomer, since the presence of LAs is paradoxically associated with thrombosis. Among the aPLs , LA is associated with the highest risk of arterial thrombosis and VTE [34]. The diagnosis of APS is challenging, as there is no single test with sufficient sensitivity and specificity to confirm a specific diagnosis for this disorder. Compounding the diagnostic problem is the variable methodology and range of tests performed by different laboratories, the lack of test standardization, as well as variation in the final interpretation of results (a postanalytical issue). The panel of tests used for diagnosing LA currently include the APTT, dRVVT , kaolin clotting time (KCT), and silica clotting time (SCT ).

    Treatment of Thrombosis

    Treatment or prevention of thrombosis include

    • Unfractionated Heparin (UFH)
    • Low Molecular Weight Heparin (LMWH)
    • Vitamin K Antagonist (VKA)
    • Direct Oral Anticoagulants (DOACs)
    • Dilute Thrombin Time (DTT)
    • Anti Xa Assays

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