BSI: Hemostasis in Hematology

Hemostasis is vital for life and is achieved by a delicate balance between procoagulants and anticoagulants.

Hemostasis is blood clotting or coagulation as a mechanism to prevent significant blood loss from the circulation.

Hemostasis is the prevention of blood loss when a vessel is severed or ruptured.

Hemostasis utilizes several mechanisms to limit blood loss until the vessel is repaired including vascular constriction, formation of a platelet plug, formation of a blood clot, clot retraction and finally removal of the clot (dissolved) after repair.

Vascular constriction attempts to limit blood loss immediately.

● Immediately after a blood vessel is severed or ruptured, the trauma (and possibly the stretch: unitary smooth muscle in blood vessel walls) causes the vessel to constrict to limit blood loss.

● This vasoconstriction is primarily caused by local factors released from the damaged tissue, Thromboxane A2 released from platelets (TXA2 synthesized from arachidonic acid formed by PLA2), and nervous reflexes initiated principally by pain receptors.

● Platelets adhere to the damaged endothelium (simple squamous epithelial cells lining/forming all blood vessels) and release TXA2 causing vasoconstriction whereas adjacent undamaged endothelium releases Prostaglandin I2 (PGI2: also called Prostacyclin and also synthesized from arachidonic acid formed by PLA2) which inhibits platelet aggregation from spreading inappropriately.

Platelets or thrombocytes are very important cell fragments vital to hemostasis.

Platelet plugs may be sufficient for minor leaks.

● Leaks occur frequently in the smaller vessels and a platelet plug is often sufficient to seal them: blood coagulation is not necessary.

• ● Platelets (also called Thrombocytes) are cell fragments ~1 - 4m in diameter formed from Megakaryocytes (see previously) in the
• bone marrow or blood especially as they squeeze through the smaller capillaries.

● Normally blood contains ~150,000 - 300,000 platelets/mm³ (mL). Platelets have no nucleus and therefore like RBCs cannot replicate.

● Platelets contain contractile proteins including actin and myosin (as found in muscle) and thrombosthenin which allow the platelets to rapidly release intracellular granules as necessary.

• ● Platelets also contain remnants of the ER and Golgi apparatus in order to:
• produce enzymes
• store Ca²⁺
• produce mitochondria and the enzymes necessary to produce ATP
• produce prostaglandins
• produce two important proteins (namely Fibrin-stabilizing factor and a growth factor which stimulates endothelial cells, vascular smooth muscle cells and fibroblasts to multiply and grow...all of which are necessary for repair)

● On the external face of the platelet cell membrane are glycoproteins which prevent them from adhering to the normal undamaged endothelial surface but does adhere to damaged endothelium and even more so to exposed collagen fibers from the underlying connective tissue.

● Platelets also contain high concentrations of phospholipids that are involved in blood clotting.

● Platelets have a short ½-life (~8 - 12 days) and are removed by macrophages in the spleen (phagocytosis).

● Contact between platelets and damaged endothelium or exposed collagen stimulates the platelets to swell and assume irregular shapes and radiating pseudopodia: contractile proteins cause the release of intracellular granules containing important factors. The platelets become "sticky" and adhere to collagen and a protein called von Willlebrand factor from blood. In addition they secrete large amounts of ADP (blocked by "Plavix") and TXA2 which activate additional nearby platelets which themselves then adhere to the original ones in a + feedback loop: this is the platelet plug.

● The plug is loose at first and this is often adequate to plug the leak but subsequently during blood clotting the fibrin fibers formed further strengthen it.

● A blood clot (as distinct from a platelet plug) starts to form after ~15 - 20 seconds if trauma is significant and after ~1 - 2 minutes if minor. This process is initiated by substances released from the damaged endothelium, platelets and blood proteins adhering to the damaged blood vessel lining.

• ● After ~3 - 6 minutes most reasonable holes are plugged by the clot. After ~20 - 60 minutes the clot
• retracts due to platelets and closes the hole even more and more tightly.

• ● Once a clot has formed there are two options:
• 1) the clot can be invaded by fibroblasts (stimulated by the platelet growth factor) which form fibrous connective tissue throughout the clot; or
• 2) the clot can be dissolved if appropriate.

• ● If invaded by fibroblasts the repair is usually complete (reorganized into fibrous connective
• tissue) after ~1 - 2 weeks.

● Blood clotting is a complex process primarily involving two overlapping enzyme cascades that usually occur in the blood but that can also occur in tissues if necessary.

● There are >50 substances found to affect or cause clotting. Some promote clotting ("procoagulants") while others inhibit clotting ("anticoagulants"). There is a balance between these two groups of substances. Normally anticoagulants dominate but when there is damage, procoagulants dominate at the site.

• ● The process can be simplified to three essential steps:
• 1) trauma stimulating either of two enzyme
• cascades resulting in the formation of a complex called Prothrombin activator;
• 2) The subsequent conversion of Prothrombin into the active enzyme Thrombin;
• 3) The conversion of the soluble plasma protein Fibrinogen into insoluble Fibrin.

● Fibrin fibers enmesh platelets, RBC's and plasma to form the clot.

Fibrinogen

Prothrombin

• Tissue Factor
• Tissue Thromboplastin

Calcium

• Proaccelerin
• Labile Factor
• Ac-globulin (Ac-G)

• Serum prothrombin conversion accelerator (SPCA)
• Proconvertin
• Stable Factor

• Antihemophilic factor (AHF)
• Antihemophilic globulin (AHG)
• Antihemophilic Factor A

• Plasma thromboplastin component (PTC)
• Christmas Factor
• Antihemophilic Factor B

• Stuart Factor
• Stuart-Prower Factor

• Plasma thromboplastin antecedent (PTA)
• antihemophilic factor C

Hageman factor

Fibrin-stablizing Factor

Fletcher Factor

Fitzgerald factor

Release Thromboxane A2 (TXA2)

● Prothrombin activator is formed by two different enzyme cascades: the "Extrinsic" pathway is principally initiated by trauma to the endothelium and surrounding/supporting tissue while the "Intrinsic" pathway originates in the blood itself.



● Both pathways utilize plasma proteins called "clotting factors" which are mostly proteolytic enzymes which act in a cascade activating subsequent members similarly to the MAPKKK signal transduction pathway (those were kinases however). The factors are designated by Roman numerals (like the cranial nerves) and the "active" form is distinguished by an "a."

● The Extrinsic pathway is initiated by the release of Tissue factor (factor III: also called Tissue Thromboplastin) which is composed primarily of phospholipids from the membranes of damaged cells.

● This complexes with and activates factor VII (→VIIa) and together with Ca²⁺ activates factor X (→Xa).

● Factor Xa combines with phospholipids from Tissue factor or platelets plus factor V to form prothrombin activator; initially however factor V is inactive in this complex.

• ● Again in the presence of Ca²⁺ within a few seconds prothrombin is converted to active
• thrombin: factor V is now activated by thrombin in a + feedback loop which in turn increases the activity of factor Xa, etc..

• ● The Intrinsic pathway is initiated by trauma to blood itself or exposure to collagen: platelets are
• activated and release phospholipids (including the lipoprotein Platelet factor III [not Tissue factor III]) and factor XII is activated (→XIIa).

● This starts a cascade whereby XIIa together with High Molecular Weight Kininogen (and accelerated by Prekallikrein) activates XI (→XIa) which in turn activates IX (→IXa) in the presence of Ca²⁺.

● IXa together with activated factor VIII (→VIIIa: increasingly activated by thrombin so forming another + feedback loop analogous to that seen previously in the Extrinsic pathway with factor V), Ca²⁺ and platelet phospholipids (including Platelet factor III) activate factor X (→Xa).

• ● This pathway is blocked if deficient in either factor VIII or platelets: a deficiency in factor VIII is
• the prime cause of “classic” hemophilia while if due to ↓no. platelets it is termed Thrombocytopenia.

● The remainder of this pathway is the same as for the Extrinsic pathway with the formation of Prothrombin activator.

● As can be seen Ca²⁺ is involved in several places in both pathways. The absence of Ca²⁺ therefore prevents blood clotting by either pathway and its removal can prevent clotting in blood samples (deionizing, combining with citrate or oxalate [precipitates Ca²⁺]).

• ● Rupturing a blood vessel will activate both
• pathways simultaneously: Tissue factor (factor III) released from damaged tissue will activate the Extrinsic pathway while exposed collagen from the
• underlying connective tissue will activate the Intrinsic pathway.

● However the Extrinsic pathway can proceed very rapidly especially with the release of  amounts of Tissue factor ( response with  damage): clotting can begin within ~15 sec!

● The Intrinsic pathway is generally much slower typically taking ~1-6 minutes to form a clot.

Blood clotting is initiated by a variety of stimuli and proceeds via two pathways (the intrinsic and the extrinsic) that share common factors and an end point: the formation of fibrin.

Ca²⁺ is vital to initiating the pathways that lead to blood coagulation.

● Any leak of blood from the vasculature must be prevented, but inappropriate clotting must also be prevented. A delicate balance must be achieved whereby clotting is initiated rapidly and effectively where it is needed (Procoagulants prevail) but not elsewhere (anticoagulants prevail).

• ● Endothelial Surface Factors are probably the most important in preventing inappropriate clotting. For example, these include:
• 1) the “smoothness” of the endothelial surface which prevents activation of the Intrinsic pathway;
• 2) the glycocalyx covering which repels clotting factors and platelet attachment/aggregation and a protein expressed on the endothelial surface
• called Thrombomodulin which binds to Thrombin and inhibits its function.

● In addition a protein activated by this complex (Thrombomodulin + Thrombin) called Protein C acts as an anticoagulant by inactivating factors Va and VIIIa.

● These inhibiting factors (anticoagulants) are all lost or reduced when the endothelial surface is compromised as occurs when an athersclerotic plaque is formed.

Normally, clotting is prevented by endogenous anticoagulants.

If repair does occur, then clots are normally removed by plasmin.

• ● Some important anticoagulants are found in the blood itself (endogenous) and act by inactivating or removing Thrombin. The most powerful of these are the fibrin fibers themselves (the “end product” of the
• clotting mechanisms) and an a-globulin called Antithrombin III (also called Antithrombin-heparin Cofactor).

● As a clot forms ~85-90% of the active Thrombin formed is adsorbed onto fibrin fibers so inactivating it which helps limit the spread of the clotting process. Any remaining Thrombin combines with Antithrombin III which also inactivates over ~12-20 minutes.

● Heparin is a powerful endogenous anticoagulant that is normally present in the blood and tissues but in amounts ↓level that it is ineffective. It is administered in  doses clinically to prevent intravascular clotting.

● Heparin itself is a virtually inactive conjugated polysaccharide that when combined in Antithrombin III increases its activity ~x100-1000.

● This complex (Heparin + Antithrombin III) also removes activated factors IXa, Xa, XIa and XIIa.

● Heparin is secreted by many cell types but especially by Mast cells in connective tissue and Basophils in blood.

● Many Mast cells are located near the lungs and liver where the slow “percolation” [Pharmacology definition of percolation: the extraction of the soluble principles of a crude drug by the passage of a suitable liquid through it] of blood can cause the formation of many potentially dangerous small emboli (unattached blood clots circulating which can block blood vessels).

• ● Plasma contains a protein called Plasminogen (also called Profibrinolysin) which when activated
• forms Plasmin (or Fibrinolysin) which is a proteolytic enzyme similar to the digestive
• enzyme Trypsin: when active it “digests” fibrin fibers, Fibrinogen, Prothrombin and factors V, VIII and XII.

• ● When a clot forms,  amounts of inactive Plasminogen are trapped and require activation by Tissue Plasminogen Activator (t-PA)
• which is released very slowly (over several days) from the damaged tissue. This therefore delays the onset of clot removal until the necessary repairs have occurred.

● Excessive bleeding can result from a deficiency of any of the previously discussed Procoagulants.

• ● There are three conditions that are particularly significant in being able to cause excessive bleeding, and they are:
• 1) vitamin K deficiency
• 2) hemophilia
• 3) thrombocytopenia

• 1) Vitamin K Deficiency
• 2) Hemophilia and Factor VIII
• 3) Thrombocytopenia (platelet deficiencies)

● Almost all the clotting factors (Procoagulants) are synthesized in the liver. Therefore, any liver disease can affect the production of procoagulants resulting in an  tendency to bleed.

• ● Vitamin K is necessary for the synthesis of four important clotting factors including Prothrombin and
• factors VII, IX and X. Production of the anticoagulant Protein C is also affected.

● Vitamin K is normally synthesized by symbiotic gut bacteria and is therefore not a limiting factor (neonates are still establishing normal gut “fauna and flora” and therefore are an exception to the rule...they frequently receive Vitamin K injections after birth to make up for this lack of production for the first week till their guts kick in).

● Because Vitamin K is fat soluble, anything interfering with this (this what, Dr. Wright?) can result in a deficiency (absence/↓amounts of bile can cause this). Therefore, any patient scheduled for surgery with liver problems is given vitamin K 4-8 hours prior to surgery to ensure adequate production of clotting factors.

● Vitamin K₁ is also known as phylloquinone or phytomenadione (also called phytonadione). Vitamin K2 (menaquinone, menatetrenone) is normally produced by bacteria in the large intestine, and dietary deficiency is extremely rare unless the intestines are heavily damaged, are unable to absorb the molecule, or are subject to decreased production by normal flora, as seen in broad spectrum antibiotic use. [Wikipedia 1/13/2011]

• ● Hemophilia is primarily genetic/inherited. It is a “sex-linked” disease (X-chromosome: ♀ carriers vs. ♂ expressed) is almost exclusively
• found in males. ~85% of cases are due to a deficiency or abnormality of Factor VIII and occurs at a frequency ~1 in 10,000 in U.S. males; this is “classic” hemophilia.

● The remaining ~15% of cases are primarily due to factor IX deficiency (also “sex-linked” so also found almost exclusively in ♂s).

● In some extreme cases a slight trauma, such as loosing a tooth, can result in several days of bleeding!

• ● Factor VIII has two active parts:
• 1) the smaller component of Factor VIII is very important for the Intrinsic pathway, which is deficient in “classic” hemophilia;
• 2) the larger component of Factor VIII is deficient in von Willebrand’s Disease which has different characteristics.

• ● The only real treatment for the previous two conditions is to infuse human-sourced
• Factor VIII which is very expensive.

● Platelets are normally present in the blood in numbers of 150,000-300,000/mL. Problems can occur if this falls below 50,000/mL and can be lethal if <10,000/mL.

● ↓no. platelets tends to result in many tiny leaks not being repaired effectively (because a “platelet plug” is not formed). Superficially, this can result purplish blotches in the skin (called “purpura”) primarily due to the loss of clot retraction by platelets.

● The most common condition of this type is called idiopathic thrombocytopenia (meaning thrombocytopenia of unknown cause) which seems to be the result of autoimmune platelet destruction by antibodies.

● Can be treated by infusion of whole blood and/or a spleenectomy. Removing the spleen would stop the removal of platelets.

Inappropriate clot formation, also known as a thromboembolic condition, can be prevented by anticoagulants and removed by t-PA.

● Abnormal or inappropriate clotting in a blood vessel is called a thrombus (multiple are called thrombi). If the thrombus or a portion of the thrombus breaks loose, it is called an embolus (multiple are called emboli) and can move through the circulation until it blocks a narrower vessel.

• ● There are two main causes for this inappropriate clotting:
• 1) any roughened endothelial surface such as that
• caused by an arteriosclerotic plaque can initiate clotting
• 2) very slowly moving blood which allows the accumulation of procoagulants which are constantly being formed.

● Current treatment involves perfusing genetically-engineered t-PA which activates Plasmin to dissolve the clot.

• ● Bed-ridden or immobile patients are particularly at risk (or those not moving for long periods of time, as in travel) as large clots can occur in leg veins from which emboli can break free and enter the right-side of the heart and onto the lungs where they can block the pulmonary arteries (pulmonary embolism). If both pulmonary arteries are blocked simultaneously
• then death will occur rapidly.

• ● If a patient has  amounts of traumatized or necrotic tissue, this can result in  amounts of
• tissue factor being released into the blood where it can cause widespread clotting called Disseminated
• Intravascular Coagulation.

● Sometimes it is important to delay the clotting process by using anticoagulants. The most useful of these are Heparin and the Coumarins such as Warfarin.

• ● Heparin can delay clotting time up to ~30 min. and the effect is immediate. Heparin is inactivated by an enzyme in the blood called Heparinase
• after ~1.5 to 4 hours.

● The Coumarins like Warfarin cause ↓plasma levels of Prothrombin and factors VII, IX and X which are all synthesized in the liver. They act by competing with vitamin K.

• ● Thrombin antagonists/inhibitors are also being investigated (such as Hirudin from the medicinal leech) as are thrombin agonists selective for it's
• anticoagulant properties.

● The clotting time of patients is routinely checked in hospital laboratories if any problem is suspected.

● Prothrombin levels are estimated by measuring the clotting time in the presence of excess Ca²⁺ and Tissue factor. Other factors are estimated in a similar manner with all components present in excess (so not limiting) except for the factor being estimated.

Thromboxane A2 (TXA2), which is formed via PLA2/arachadonic acid pathway.

The stretching of unitary smooth muscle in blood vessel walls and neurogenic reflexes initiated by pain receptors.

• Factor V
• Factor X
• platelet phospholipids
• Ca²⁺

Initially it is inactive, but a positive feedback loop from Thrombin increasingly activates it.

• Primarily converts fibrinogen to fibrin
• Creates positive feedback loop in Factor V, Factor VIII, and Factor XI
• Acts as an anticoagulant linking Protein C to Thrombomodulin on endothelial cells, which then degrades Factors V and Factor VIII

• Hemostasis is vital for life and is achieved by a delicate balance between pro- and anticoagulants.

• Vascular constriction attempts to limit blood loss immediately.

• Platelets (thrombocytes) are very important cell fragments vital to hemostasis.

• Platelet plugs may be sufficient for minor leaks.

• Blood clotting is initiated by a variety of stimuli and proceeds via two pathways that share common factors and an end point: the formation of fibrin.

• Without Ca²⁺ you won't have blood coagulation. Ca²⁺ is a vital part of these pathways and processes for blood clotting.

•Normally clotting is prevented by endogenous factors such as heparin (anticoagulants).

•After repair, clots are normally removed by plasmin.

•Vitamin K and platelet deficiencies plus inherited clotting factor abnormalities can all cause excessive bleeding (hemophilia).

•Inappropriate clot formation (thromboembolic conditions) can be prevented by anticoagulants and removed by t-PA.