What is hemostasis?
What is hemostasis followed by?
What are the 3 phases of hemostasis?
Hemostasis (greek haimóstasis = antihemorrhagic process) is the process that prevents excessive loss of blood following injury of the blood vessels. This is followed by fibrinolysis; the removal of the clot after the blood vessel is healed.
Phase 1: Procoagulation Phase
2: Anticoagulation Phase
What two things must occur for blood clotting to happen? (procoagulation phase)
Blood clotting occurs by vessel constriction followed by platelet aggregation (cellular aspect) and activation of the plasma blood coagulation system (molecular or enzymatic aspect).
What is the role of vasoconstriction in damage to a blood vessel? What stimulates vasoconstriction in this instance?
Vasoconstriction: Damaged vessels constrict caused by vasoconstrictive paracrine released by the endothelium. Vasoconstriction temporarily decreases blood flow and pressure within the vessel.
What is the initial response to damage to a blood vessel?
Platelet aggregation: The initial response to blood vessel damage is platelet aggregation (part of procoagulation phase). Platelets are small, non-nucleated blood cells that circulate in the blood and aggregate at the site of damage triggered by several factors.
Normally (when not injured), what do endothelial cells have on their cell surface to inhibit coagulation? What do they secrete to inhibit coagulation?
After endothelial cell injury, what do endothelial cells release?
Explain how platelet aggregation at the site of injury is initiated.
Normal endothelial cells: prevent platelet attachment They have thrombomodulin on their cell surface (anticoagulant) They release nitric oxide and prostacyclin (PGI2) (a prostaglandin derivative-COX pathway)
Injured endothelial cells release the von Willebrand factor (vWF), thromboxane and ADP.
Sub-endothelial collagen of the intima is exposed and platelets bind to it through the vWF. Then, platelets are activated and release thromboxane (TXA2, a prostaglandin derivative) and ADP (among other things). All of the above promote platelet adhesion and aggregation.
What processes are facilitated by factors secreted by platelets? Other than TXA2, vWF, and ADP, what else is secreted by platelets and what are their functions?
Serotonin is released: stimulates vasoconstriction
Growth factors: to immediately stimulate healing process
Coagulation factors: part of molecular aspect of blood clotting
attached is slide 16 of pp
What is aggregometry? How does it work?
What is a normal test?
Precipitation of platelets after addition of ADP. Platelet rich human blood plasma (left vial) is a turbid liquid. Upon addition of ADP, platelets are activated and start to aggregate, forming white flakes (right vial).
Normal pattern: 80-90% of platelets should aggregate.
What is the goal of the molecular aspect of blood clotting?
What enzyme is key for this to occur?
In what form do the enzymes that are involved in this process circulate in? (active, inactive)
The purpose of the plasma molecular blood clotting system is the enzymatic conversion of fibrinogen (Factor I) into an insoluble fibrous fibrin polymer. The enzyme that catalyzes this reaction is thrombin (Factor II). All the enzymes before this step are there to ensure that the required number of active thrombin molecules is present at the site of damage.
The cascade is a series of enzymatic reactions that ends in the formation of a fibrin protein fiber mesh that stabilizes the platelet plug.
The enzymes circulate in blood in zymogen (proenzyme) form (labeled by F and a roman numeral, e.g. FII). Proteolytic cleavage will activate these proteinases (labeled by “F”, the factor’s number followed by “a”, e.g. FIIa)
What class of enzymes are the enzymes involved in the molecular aspect of blood clotting?
Explain the instrinsic, extrinsic, and common pathways of the molecular aspect of blood clotting.
All of these factors (except for FV and FVIII) are serine
proteinases. All of these factors circulate in zymogen forms. They activate each other in a well-organized system, by cleaving the propeptides off and turning the enzyme to active form.
Intrinsic pathway is triggered by the exposure of anionic membrane surfaces of the subendothelial cells and of collagen. FXII binds to this new surfaces and activates kallikrein, which in turn will cleave factor XII and produces FXIIa, the active enzyme. This active enzyme will cleave FXI producing FXIa, which then will activate FIX to FIXa. This enzyme, in the presence of FVIIIa (this is a regulatory protein, not an enzyme) will cleave FX to FXa. This is a 4-step cascade.
The extrinsic pathway is initiated by tissue factor (TF or FIII) being exposed by injury on the surface of the sub-endothelial cells. FVII will bind TF and being activated. FVIIa then can activate FX to FXa. This is a 2-step cascade.
Common pathway: The two pathways join at FX. Active FXa will form a complex with FV (a regulatory protein, not an enzyme) and activate prothrombin (FII) to thrombin (FIIa). Thrombin can then cleave fibrinogen, and also help activating Factors V, VII, VIII and XIII, to speed up the blood coagulation process.
What is the function of fibrin? What is the function of FXIII?
Thrombin then proteolytically cleaves some peptides off of fibrinogen (FI) and the resulting fibrin molecules can aggregate, forming a network intertwined with the platelet aggregate to block the damage site of the blood vessel. Please note that fibrinogen is not an enzyme but a fibrillar protein.
The aggregated fibrin network then will be stabilized by crosslinking by FXIII, activated by thrombin to XIIIa, also called transglutaminase, which is not a serine proteinase, but a ligase. This forms the hard clot.
Note that thrombin also functions in attraction of platelets.
What is Gla (gamma carboxy glutamate)? What coagulation factors have Gla?
What is the importance of Gla residues for coagulation?
What is the importance of vitamin K in coagulation?
Gla is gamma carboxy glutamate, a Glu residue in proteins that has 2 carboxy groups atthe end of its side-chain. Gla is formed by posttranslational modification of Glu side chains in several coagulation factors, including factors II, VII, IX, X and Protein C.
Gla is important in Ca ion binding, thus coagulation factors can bind to phospholipids of the membranes of platelets through a Ca-bridge. Consequently, the coagulation system is localized strictly to the site of injury. Moreover, when Gla-containing factors are activated, the Gla peptide that is cleaved off from the factors during activation goes to the liver and stimulates the synthesis of coagulation factors.
Vitamin K is necessary for the formation of Gla from Glu. Deficiency of Vitamin K, thus, results in bleeding disorder, since factors are not modified to Gla and cannot be secreted from the liver.
Explain the factors and processes involved in phase 2, anticoagulation.
Control of blood clotting:
1. Several inhibitors circulate in the plasma that regulate the extent of blood clotting. These are serpins, serine proteinase inhibitors. Most important is antithrombin III (needs heparin for activity), which inhibits all serine proteinases, most specifically thrombin and FXa. The extrinsic pathway is inhibited by anticonvertin (tissue factor inhibitor, TFPI).
2. Thrombin together with another protein (protein S, PS) activates Protein C, an enzyme that degrades FV and FVIII, the two regulatory proteins, without which FX and FIX will not work.
Explain what enzymes are involved in fibrinolysis. State where these enzymes are made and their functions.
Fibrin networks should be degraded after healing of the blood vessel. The enzyme is plasmin, again, a serine proteinase, which circulates in zymogen form (plasminogen).
Other proteinases activate plasminogen: urokinase, which is formed in kidney and released in response to injury, and tissue plasminogen activator (t-PA) produced by the healed vascular endothelial cells.
What inhibits the fibrinolysis pathway?
What inhibits the action of plasmin?
This pathway will be inhibited by plasminogen activator inhibitors and plasmin action will be regulated by a2 –antiplasmin inhibitor.
Explain the affects of the following drugs on coagulation
Both prostaglandins (PGI2) and thromboxanes (TXA2) are synthesized by the cyclooxygenase (COX) pathway. While PGI2 is anti-thrombotic, TXA2 is pro thrombotic. COX inhibitors thus interfere with normal hemostasis, often times inhibit coagulation. These are steroidal and non-steroidal anti-inflammatory drugs (aspirin, ibuprofen and indomethacin) that may cause bleeding.
Platelet aggregation induced by ADP is inhibited by Plavix (clopidogrel).
Vitamin K analogs, dicoumarol and warfarin, inhibit Gla formation, thus clotting. (binds to same site as Vit K but inhibits Gla formation)
Xarelto is a Factor Xa inhibitor.
What are the 3 most common types of inherited bleeding disorders? What are they caused by?
In comparison to inherited bleeding disorders, how common are acquired bleeding disorders? Give examples of acquired bleeding disorders.
The most common are Hemophilia A or classic hemophilia (factor VIII deficiency), Hemophilia B or Christmas disease (factor IX deficiency) and von Willebrand disease (decreased vWF)
Acquired bleeding disorders: are varied and occur more frequently than inherited disorders. common ones include multiple factor deficiencies caused by liver disease or vitamin K deficiency, side-effect of certain drugs
What are the inheritance patterns for hemophilia A and B?
How are they treated?
Which is more prevalent?
1. Classic Hemophilia - Hemophilia A
- X-linked recessive disorder
- 1 in 5,000 males is born with a deficiency of factor VIII.
- Deficiency of factor VIII. (<1% severe; 1-5% moderate; >5% mild).
- Treatment: blood transfusion (possibility of hepatitis or HIV, autoantibodies)
- recombinant factor VIII
- Of the approximate 25,000 hemophiliacs in the United States, more than 80% are of the A type.
2. Christmas disease – Hemophilia B
- X-linked recessive disorder
- 1 in 35,000 males is born with a deficiency of factor IX.
- deficiency of factor IX (pointmutation).
- Treatment: factor IX replacement.
How common is von Willebrand disease (as is pertains to congenital bleeding disorders)?
How is von Willebrand disease treated?
How can one acquire inadequate platelet function?
The most common congenital bleeding disorder is von Willebrand disease, characterized by low levels of circulating vWf. Endothelial cells release vWf; if compromised, platelets do not attach to intima collagen. A drug, Desmopressin is used to stimulate endothelial cells to release vWf.
Acquired disorder can result in underproduction of platelets by bone marrow cells or excessive destruction of platelets by autoimmune process.
What pathways does PT (prothrombin time) test? How is PT assessed?
What pathways does APTT (activated partial thromboplastin time) test? How is APTT assessed?
What does thrombin time (TT) test? How is TT assessed?
Prothrombin time (PT): to test the extrinsic and common pathways.
- Tissue factor (TF) and CaCl2 are added to the plasma and normal clotting time is measuredif all factors work well in the extrinsic and common pathways
- Normal clotting time is 10-15 sec.
Activated partial thromboplastin time (APTT): to test the intrinsic and common pathways
- Kaolin (activating surface) and brain cephalin (to stimulate platelet factors) are added to the plasma. Normal clotting time is measured if all factors work well in the intrinsic and common pathways.
- Normal clotting time is 30-50 sec.
Thrombin time (TT): to test fibrinogen deficiency; or the presence of an inhibitor to fibrinogen conversion to fibrin
- Thrombin is added to plasma – since this enzyme is responsible for activating fibrinogen to fibrin, normal clotting time is measured only if fibrinogen is present in normal concentration in the plasma.
- Normal clotting time is 10-15 sec.
Note: Tests are also available to detect coagulation factor inhibitors.
What can be done for the patients?
Identify, monitor, control.
Treatment is dependent on the cause and severity of the condition.
Treatments may include the following:
- avoid injury, limit physical contacts in daily environment
- replacement of coagulation factors (FVIII and a few others are available in a concentrated form)
- transfusions of fresh frozen plasma or plasma concentrates
- platelet transfusion
- desmopressin therapy (for von Willebrand disease)
- vitamin K supplementation (for vitamin K deficiency)
- immune suppressive drugs (if antibodies are present)